Methods and compositions using coiled binding partners

ABSTRACT

The present invention relates to a polypeptide multimer comprising a first polypeptide having associated therewith a label and a second polypeptide, wherein a) at least one of the polypeptides is susceptible to protease digestion; b) association of the polypeptides to form a multimer is detectable via a signal emitted by the label; and c) digestion of at least one polypeptide results in dissociation of the multimer and modulation of the signal emitted by the label, and the method of detecting or monitoring the activity of a protease enzyme based on such a multimer.

FIELD OF THE INVENTION

The present invention relates to a method for detecting or monitoringthe activity of an enzyme. In particular, the invention relates the useof a polypeptide multimer capable of generating amultimerisation-dependent signal and whose multimerisation propertiesare modulated by the activity of a protease in such a method.

BACKGROUND

Proteolysis has long been recognised as an important intra- andextracellular modification of proteins. Endopeptidase enzymes recogniseparticular primary sequence signals (and sometimes also secondary ortertiary structural cues) within a substrate protein and cleave thepeptide bond following a particular amino acid. Exopeptidases, on theother hand, digest polypeptides from the N or C terminus. Exopeptidasesare generally not sequence-specific. These enzymes play a role in, forexample, digestion, the coagulation of blood, the complement cascade andthe destruction of inactive, mutated or foreign forms of proteins in thecell. Proteolysis is also important as a method of, recycling aminoacids within the cell for the synthesis of new proteins or forutilisation as a fuel source. More recently, the role of proteolysis insignalling and in specific intracellular processes has been recognised.

It is clear that aberrant proteolysis plays a significant role in anumber of disease processes. Examples include the processing ofβ-amyloid precursor protein (inappropriate processing of this protein isthought to play a role in Alzheimer's Disease), the inappropriateactivation of proteolytic enzymes of digestion leading to pancreatitisand a loss of proteolysis of the insulin receptor precursor leading todiabetes. Proteolysis is now understood to play important roles bothwithin the cell and in processes important in homeostasis inmulti-cellular organisms. These include:

Production of bioactive molecules from inactive precursors. A hallmarkof proteolytic enzymes is their production in many cases as inactiveproenzymes and their subsequent rapid activation by a proteolytic event.This may be an autocatalytic process or part of a cascade. This isexemplified by the blood clotting cascade and also the cleavage ofdigestive proproteases to their active form. One of the central eventsin acute pancreatitis is the premature proteolysis and activation ofpancreatic enzymes (especially trypsin) leading to autodigestion ofpancreatic tissue amongst other effects (Acute pancreatitis, Mergener, K& Baillie, J. British Medical Journal (1998) 316 44-48). Proteases arealso known to activate other proenzymes and to play a role in thegeneration of other bioactive molecules. An important clinical exampleof this is the generation of angiotensin II by the enzyme angiotensinconverting enzyme (ACE). ACE cleaves the C-terminal two residues fromthe inactive angiotensin I to produce the active form, angiotensin II.Angiotensin II has potent vasoconstrictive and salt-retentiveproperties, the control of ACE activity by ACE inhibitors has animportant clinical role in the treatment of hypertension, heart failure,myocardial infarction and diabetic nephropathy (Angiotensin convertingenzyme inhibitors, Brown NJ. & Vaughan, DE., Circulation (1998) 971411-1420).

Destruction of bioactive molecules. An important aspect of a regulatoryprocess is the presence not only of an ‘on switch’ but also thepotential to switch it off again. This is an area in which proteolysisis particularly important as it is an irreversible modification. Theonly way in which the process can be restarted is by a resynthesis ofthe destroyed component. This affords a high level of control overtiming. An important clinical example of this is the degradation ofbradykinin by ACE. Bradykinin has a number of effects in the bodyincluding inducing smooth muscle contraction, increasing vascularpermeability and promoting vasodilation and natriuresis. This, togetherwith the example above, indicates that ACE is important in theregulation of the balance between the antagonistic effects ofangiotensin II and bradykinin (Angiotensin converting enzyme inhibitors,Brown NJ. & Vaughan, DE., Circulation (1998) 97 1411-1420).

Protein turnover. The ability of the cell to degrade unwanted, damagedor foreign proteins is of great importance in the maintenance of thecell. Limited proteolysis of foreign proteins is also important in theantigen presentation process and therefore in an appropriate immuneresponse to pathogens.

Post-translational modification. The proteolysis of certain proteins iskey in their ability to perform their function in the cell. For example,the biosynthesis of the insulin receptor involves the cleavage of alarge precursor to produce the subunits of the receptor complex(Biosynthesis and glycosylation of the insulin receptor, Hedo, J. A.,Kahn, C. R., Hayashi, M., Yamada, K. M., Kasuga, M., Journal ofBiological Chemistry (1983) 258 10020-10026). The assembly of the plantlectin concanavalin A (con A) also involves the proteolysis of aprecursor protein and the religation of fragments in an altered order togenerate the mature protein (Traffic and assembly of concanavalin A,Bowles, D. J. & Pappin, D. J., Trends in Biochemical Science (1988) 1360-64).

A process coincident with other forms of post-translationalmodification. Proteolysis is an important feature of the processesleading to the addition of glycosylphosphatidylinositol (GPI) anchors toproteins and also in some fatty acylation reactions (such asfarnesylation or geranylgeranylation).

Thus, proteolysis is an important post-translational modification ofproteins and peptides which occurs both within and outside of the celland can be an essential part of other forms of post-translationalmodification such as addition of a GPI anchor or some fatty acids. Theability to measure the cleavage of a protein or peptide at a specificsite where that protein or peptide is also accessible for the additionof a prenyl moiety or a GPI anchor will allow the in vitro and in vivostudy of processes for which the methods currently available arelimited.

However, methods presently available for monitoring or detectingprotease activity are not sufficiently sophisticated to be useful.Reporters are currently available to follow proteolysis where a peptidecontaining the cut site of the protease of interest has fluorophores ateither end. Modification is followed by a change in the fluorescentoutput on cleavage of the peptide (causing physical separation of thefluorophores). Methods are available for monitoring both in vivo and invitro proteolysis given the availability of various chemicalfluorophores and quenchers and also a number of GFP variants which canbe expressed in the cell (Compositions for the detection of proteases inbiological samples and methods of use thereof, Komoriya, A. & Packard,B. S., WO96/13607; Tandem fluorescent protein constructs, Tsien, R. Y.,Heim, R. & Cubitt, A., WO97/28261). All such methods, however, rely onthe use of a synthetic reporter which is typically not the naturalsubstrate for the enzyme being assayed. Moreover, the flexibility of theprior art systems is limited.

SUMMARY OF TE INVENTION

According to a first aspect of the present invention, there is provideda polypeptide multimer comprising a first polypeptide and a secondpolypeptide, wherein

-   -   a) at least one of the polypeptides is susceptible to protease        digestion;    -   b) association of the polypeptides to form a multimer is        detectable via a signal; and    -   c) digestion of at least one polypeptide results in modulation        of the association state of the multimer and modulation of the        signal.

The invention accordingly provides a polypeptide multimer, or aconstituent polypeptide thereof, which is susceptible to proteasedigestion such that digestion leads to dissociation of the constituentpolypeptides, or a part thereof, from the multimer. The dissociation andassociation of the polypeptides in the multimer is in turn detectable,for example via a label (further described-below), or by monitoring ofmolecular weight, such as by surface plasmon resonance, or by measuringthe molecular interactions of polypeptides through changes in emissionor absorbance spectra of constituent parts thereof. For example, theassociation of the multimer is detectable through the interaction oflabels placed on two or more polypeptides, which differs depending onwhether the polypeptides are multimerised or not. For example, where thelabels are fluorescent labels, fluorescence resonance energy transfer(FRET) is observable when the labels are in close proximity in amultimer. FRET is absent or otherwise modulated when the multimerdissociates.

“Modulation of the signal” refers to the capacity to either increase ordecease a measurable signal by at least 10%, 15%, 20%, 25%, 50%, 100% ormore; such increase or decrease is contingent on proteolytic cleavage ofat least one polypeptide component of a multimer.

The multimer may be a homomultimer or a heteromultimer. In the former,the polypeptide monomers are substantially identical, whilst in thelatter they differ. Preferably, the multimer is a heteromultimer. In thecontext of the present invention, a “multimer” may be a dimer,consisting of two monomers; however, it may optionally be a trimer,tetramer, pentamer or hexamer, composed of groups of three or moremonomers, or dimers, trimers, etc. of constituent components which arethemselves composed of individual monomer components. In all of thesesituations, the invention requires merely that the molecule (referred toas a “multimer”) should be capable of moving between an associated and adissociated state in response to digestion of a component thereof by aprotease enzyme.

According to the invention, binding of a first polypeptide to a secondpolypeptide is dependent upon protease digestion, which digestion mayoccur on one or more polypeptides.

As referred to herein, a polypeptide is “susceptible to digestion by aprotease” if it is available for cleavage by one or more proteaseenzymes in accordance with the present invention. Advantageously, thepolypeptide is susceptible to digestion by a specific protease enzyme,and preferably only susceptible to digestion by a specific proteaseenzyme. This facilitates the reduction of non-specific or backgroundproteolysis and the use of the invention to assay specific proteolyticevents.

Advantageously, digestion preferably occurs at a protease cleavablesite, which may be engineered into the one or more of thepolypeptide(s)—an “engineered site”—or may be naturally present in oneor more of the polypeptide(s)—a “natural site”. However, it is alsopossible to design one or more polypeptide(s) such that they arepotentially exposed to digestion by a protease which initiallyrecognises a site which may be distal to the polypeptide itself—such ason a further polypeptide bound to a polypeptide according to theinvention—and/or by an exoprotease enzyme which digests non-sitespecifically from the N or C terminus of the polypeptide.

The term “protease cleavable site” refers to an amino acid sequencewhich is recognised by (i.e., a recognition site for) a protease enzyme.It is contemplated that a site comprises a small number of amino acids,typically from 2 to 10, less often up to 30 amino acids, and furtherthat a site comprises fewer than the total number of amino acids presentin the polypeptide.

An engineered protease cleavable site suitable for digestion may beplaced within a polypeptide of the invention at a position such thatformation of a multimer between the isolated polypeptide and its bindingpartner is dependent upon the intactness of the site.

It is contemplated that the position at which an engineered site is toreside may initially be determined by random placement of the sitewithin the polypeptide, followed by testing by methods described hereinof the ability of the polypeptide to associate into a multimer with itsintended binding partner(s), or not, depending upon the intactness orotherwise of the site. A pair of polypeptides, of which at least onecomprises a site so placed that association of the polypeptides isdependent on cleavage at this site, is useful in the present invention.

As used herein, the term “polypeptide” refers to a polymer in which themonomers are amino acids and are joined together through peptide ordisulphide bonds. “Polypeptide” refers to a full-lengthnaturally-occurring amino acid chain or a fragment thereof, such as aselected region of the polypeptide that is of interest in a bindinginteraction, or a synthetic amino acid chain, or a combination thereof.“Fragment thereof” thus refers to an amino acid sequence that is aportion of a full-length polypeptide, between about 8 and about 500amino acids in length, preferably about 8 to about 300, more preferablyabout 8 to about 200 amino acids, and even more preferably about 10 toabout 50 or 100 amino acids in length. Additionally, amino acids otherthan naturally-occurring amino acids, for example β-alanine, phenylglycine and homoarginine, may be included. Commonly-encountered aminoacids which are not gene-encoded may also be used in the presentinvention. All of the amino acids used in the present invention may beeither the D- or L—optical isomer. The D-isomers are preferred for usein a specific context, further described below. In addition, otherpeptidomimetics are also useful, e.g. in linker sequences ofpolypeptides of the present invention (see Spatola, 1983, in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Weinstein, ed.,Marcel Dekker, New York, p. 267).

“Naturally-occurring” as used herein, as applied to a polypeptide orpolynucleotide, refers to the fact that the polypeptide orpolynucleotide can be found in nature. One such example is a polypeptideor polynucleotide sequence that is present in an organism (including avirus) that can be isolated from a source in nature. Once thepolypeptide is engineered as described herein it is no longer naturallyoccurring but is derived from a naturally occurring polypeptide.

“Polynucleotide” refers to a polymeric form of nucleotides of 2 up to1,000 bases in length, or even more, either ribonucleotides ordeoxyribonucleotides or a modified form of either type of nucleotide.The term includes single and double stranded forms of DNA. The term issynonymous with “oligonucleotide”.

As used herein, the term “associates” or “binds” refers to polypeptidesas described herein having a binding constant sufficiently strong toallow detection of binding by a detection means, such as FRET.Preferably, the polypeptides, when associated or bound, are in physicalcontact with each other and have a dissociation constant (Kd) of about10 μM or lower. The contact region may include all or parts of the twomolecules. Therefore, the terms “substantially dissociated” and“dissociated” or “substantially unbound” or “unbound” refer to theabsence or loss of contact between such regions, such that the bindingconstant is reduced by an amount which produces a discernible change ina signal compared to the bound state, including a total absence or lossof contact, such that the proteins are completely separated, as well asa partial absence or loss of contact, so that the body of the proteinsare no longer in close proximity to each other but may still be tetheredtogether or otherwise loosely attached, and thus have a dissociationconstant greater than 10 μM (Kd). In many cases, the Kd will be in themM range. The terms “complex” and, particularly, “dimer”, “trimer”,“tetramer”, “multimer” and “oligomer”, as used herein, refer to thepolypeptides, peptides, proteins, domains or subunits in the associatedor bound state. More than one molecule of each of the two or morepolypeptides may be present in a complex, dimer, multimer or oligomeraccording to the methods of the invention.

As used herein the term “modulation of the association state” refers tothe ability of a protease enzyme, as defined above, to promote, preventor reverse the association of at least two polypeptides, as definedabove, by at least 10%, preferably by 25-50%, highly preferably by75-90% and, most preferably, by 95-100% relative to the associationobserved in the absence of digestion by a protease enzyme under the sameexperimental conditions.

According to the experimental conditions, a monomer or multimer maychange its association state by partially associating or dissociatingwithout being either entirely reduced to the constituent monomerpolypeptides or present exclusively in a single multimeric form. Forexample, a single polypeptide may dissociate from a trimer, leaving apolypeptide dimer. Moreover, cleavage of one of the monomers may occursuch that the label is removed, and the signal generated on multimerformation thus modulated, but the monomer otherwise remains part of themultimer.

Alternatively, the association of polypeptides to form a multimer isinhibited by the presence of a modification which can itself be removedby proteolysis, thus allowing association of the polypeptides into themultimer. Preferably, the modification is of a residue of a coiled coilwhich is susceptible to cleavage from one of the polypeptides, such thatthe coiled coil is no longer able to form; the modification may, forexample, be a phosphorylation of one of the residues. Cleavage of thecoil such that the modified residue is removed leaves remaining coiledcoils available for association.

The “detectable signal” referred to herein may be any detectablemanifestation attributable to the presence of a label and will depend onthe means selected for label detection. For example, in the event thatthe label is detected by FRET, a label will be present on at least twopolypeptide components of the multimer such that association anddissociation thereof can be monitored by measurement of energy transferbetween the labels. However, if the label is detected for example byfluorescence correlation spectroscopy (FCS), which relies on themeasurement of the rate of diffusion of a label, only a single labelledpolypeptide is required. In the case of FCS detection, the labelledpolypeptide is advantageously very much smaller than the associatedmultimer. For example, the labelled polypeptide is preferably between 25and 50% of the molecular weight of the multimer, advantageously 10 to25%, and more preferably 1 to 10% or less.

The “label” according to the invention preferably comprises a lightemitting detection means, and the light emitting detection meansadvantageously emits light of at least a fluorescent wavelengthemission. It is preferred that the light emitting detection meanscomprises two different fluorophores or fluorescent tags or groups.

A “fluorescent tag” or “fluorescent group” refers to either afluorophore or a fluorescent protein or fluorescent fragment thereof“Fluorescent protein” refers to any protein which fluoresces whenexcited with appropriate electromagnetic radiation. This includesproteins whose amino acid sequences are either natural or engineered.

It is additionally preferred that the fluorophores comprise fluoresceinand tetramethylrhodamine or another suitable pair. In another preferredembodiment, the label comprises two different fluorescent proteins. Itis preferred that fluorescent proteins comprise any protein selectedfrom the group consisting of green fluorescent protein (GFP), bluefluorescent protein, red fluorescent protein and other engineered formsof GFP.

Preferably, the polypeptide comprises a cysteine or lysine amino acidthrough which the label is attached via a covalent bond.

Preferably, the measuring is performed by fluorescent resonance energytransfer (FRET), fluorescence anisotropy or fluorescence correlationspectroscopy.

It is preferred that the fluorescence emitting means comprise twodifferent fluorophores, and particularly preferred that the fluorophorescomprise fluorescein and tetramethylrhodamine or another suitable pair.

As used herein with regard to fluorescent labels for use in FRET, theterm “appropriate combination” refers to a choice of reporter labelssuch that the emission wavelength spectrum of one (the “donor” moiety)is within the excitation wavelength spectrum of the other (the“acceptor” moiety).

The invention also encompasses a pair of polypeptides which associate toform a multimer, the pair comprising a first polypeptide comprising atleast one binding domain, at least one site susceptible to proteolyticdigestion, and a label, whereby the proteolytic digestion of at leastone polypeptide is detectable via binding of the binding domain with asecond polypeptide; and a second polypeptide which is capable of bindingto the first polypeptide, wherein multimer formation is detectable viathe label.

The invention additionally provides a method of screening for acandidate modulator of enzymatic activity of a protease, the methodcomprising mixing in an appropriate buffer an appropriate amount of apolypeptide susceptible to protease digestion, wherein the polypeptidebinds to at least a second polypeptide, and wherein at least onepolypeptide is suitably labelled with detection means for monitoringassociation/dissociation between the polypeptides; and a sample ofmaterial whose enzymatic activity is to be tested; and monitoring thedigestion of the polypeptide.

Modulation of the association of the polypeptides to form a multimer isindicative of a modulation in the activity of the protease enzyme, andtherefore of the activity of the candidate protease enzyme modulator.

As used herein, the term “sample” refers to a collection of inorganic,organic or biochemical molecules which is either found in nature (e.g.,in a biological- or other specimen) or in an artificially-constructedgrouping, such as agents which might be found and/or mixed in alaboratory. Such a sample may be either heterogeneous or homogeneous.

As used herein, the interchangeable terms “biological specimen” and“biological sample” refer to a whole organism or a subset of itstissues, cells or component parts (e.g. body fluids, including but notlimited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinalfluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluidand semen). “Biological sample” further refers to a homogenate, lysateor extract prepared from a whole organism or a subset of its tissues,cells or component parts, or a fraction or portion thereof. Lastly,“biological sample” refers to a medium, such as a nutrient broth or gelin which an organism has been propagated, which contains cellularcomponents, such as proteins or nucleic acid molecules.

As used herein, the term “organism” refers to all cellular life-forms,such as prokaryotes and eukaryotes, as well as non-cellular, nucleicacid-containing entities, such as bacteriophage and viruses.

It is highly preferred that a method of the methods described abovecomprises real-time observation of association of an isolatedpolypeptide and its binding partner or of an isolated pair ofpolypeptides.

As used herein in reference to monitoring, measurements or observationsin assays of the invention, the term “real-time” refers to that which isperformed contemporaneously with the monitored, measured or observedevents and which yields as a result of the monitoring, measurement orobservation to one who performs it simultaneously, or effectively so,with the occurrence of a monitored, measured or observed event. Thus, a“real time” assay or measurement contains not only the measured andquantitated result, such as fluorescence, but expresses this in realtime, that is, in hours, minutes, seconds, milliseconds, nanoseconds,picoseconds, etc. Shorter times exceed the instrumentation capability;further, resolution is also limited by the folding and binding kineticsof polypeptides.

A variant of the present invention as described above involves the useof first and second polypeptide binding domains, which are located onthe same polypeptide, in a method according to the invention. Thepolypeptide is configured such that binding between the domains resultsin spatial rearrangement of labels present on the polypeptide, such thata signal is induced or modulated.

In a further aspect, the present invention provides a method formonitoring the activity of a protease enzyme, comprising the steps of:

-   -   a) providing a first binding domain having associated therewith        a label, and a second binding domain, wherein        -   i) at least one of the binding domains is susceptible to            protease digestion; and        -   ii) the first and second binding domains are capable of            binding to each other such that a detectable signal is            generated by the label, and digestion of one or both of the            polypeptides by the protease enzyme results in modulation of            the binding of the polypeptides to each other and therefore            of the detectable signal;    -   b) allowing the binding domains to bind to each other and induce        a detectable signal;    -   c) contacting the binding domains with a protease enzyme; and    -   d) detecting modulation of the detectable signal as a result of        the modulation of the binding of the binding domains.

Where more than one polypeptide is used, the polypeptides are capable ofassociating to form a multimer. Preferably the multimer is a dimer,trimer, or tetramer. Advantageously, it is a dimer. Preferably, thelabel is present on two or more polypeptide constituents of themultimer.

Where the binding domains are present on a single polypeptide, it is notnecessary for a multimer to be formed.

Detection of the signal attributable to the label in the binding domainsmay be carried out according to the invention as set forth above.

A “binding domain”, as used herein, is a polypeptide domain capable ofmediating the binding of one polypeptide to a second polypeptide.Exemplary binding domains are described below, and include bZIP domains,coiled coil domains, SH2 domains and SH3 domains.

As used herein, the term “contacting” refers to the act of placing tworeagents in such a relationship that they may potentially interact inorder to produce a chemical or biological effect. Preferably, theprocess of contacting involves admixing the reagents at an appropriateconcentration in solution or suspension, either in liquid or solidphases, or both, in an appropriate buffer.

As used herein, the term “appropriate buffer” refers to a medium whichpermits activity of the protease enzyme used in an assay of theinvention, such as a low-ionic-strength buffer or other biocompatiblesolution (e.g., water, containing one or more of physiological salt,such as simple saline, and/or a weak buffer, such as Tris or phosphate,or others as described hereinbelow), a cell culture medium, of whichmany are known in the art, or a whole or fractionated cell lysate;provided that it is compatible with the binding of the components of theassay of the invention, and with the selected signal employed. Forexample, the buffer advantageously does not include agents which quenchfluorescence, if the signal is a fluorescent signal. An “appropriatebuffer” permits digestion of polypeptides according to the inventionand, preferably, inhibits degradation and maintains biological activityof the reaction components. Inhibitors of degradation, such as nucleaseinhibitors (e.g., DEPC) are well known.

As used herein, the term “appropriate concentration” refers to an amountof reagent (for example, a labelled polypeptide of the invention) whichis sufficient for the intended reaction to proceed in a detectablemanner. For instance, in the case of a labelled polypeptide, anappropriate concentration may be considered to be that concentration atwhich the label emits a signal within the detection limits of ameasuring device used in an assay of the invention. Such an amount isgreat enough to permit detection of a signal, yet small enough that achange in signal emission is detectable (e.g., such that a signal isbelow the upper limit of the device).

Moreover, the invention relates to a method for detecting or monitoringthe activity of a modulator of a protease enzyme, comprising the stepsof:

-   -   a) providing a first binding domain, and a second binding        domain, wherein        -   i) at least one of the binding domains is susceptible to            protease digestion; and        -   ii) the first and second binding domains are capable of            binding to each other such that a detectable signal is            generated by the label, and digestion of one or both of the            polypeptides by the protease enzyme results in modulation of            the binding of the binding domains to each other and            therefore of the detectable signal;    -   b) allowing the binding domains to bind to each other and induce        a detectable signal;    -   c) contacting the binding domains with a protease enzyme;    -   d) detecting modulation of the detectable signal as a result of        the modulation of the binding of the binding domains to        determine a reference signal modulation;    -   e) contacting the binding domains with a protease enzyme and a        candidate modulator of the protease enzyme; and    -   f) detecting modulation of the detectable signal as a result of        the modulation of the binding of the binding domains, and        comparing the modulation detected with the reference signal        modulation.

A “reference signal modulation” is the amount by which a detectablesignal is modulated, as defined above, in response to the activity of aprotease enzyme in accordance with the invention. For example,therefore, the signal may be modulated, that is increased or decreased,by 10%, 15%, 20%, 25%, 50%, 100% or more. The reference signalmodulation may be calculated at any time, and used as a standard value;it need not be recalculated every time the assay is performed.Comparison of detected signal modulation values with the referencesignal modulation preferably manifest themselves as increases ordecreases in the percentage modulation with respect to the referencevalue.

The assay permits the assessment of the activity of compounds, whethernaturally-occurring or synthesised, to modulate the activity of aprotease enzyme. It thus permits the use of the invention to detect ormonitor processes which rely on protease activity or result in or fromprotease activity, such as post-translational modifications of proteins.The invention preferably relates to a method for detecting or monitoringfatty acylation of a protein, wherein the reaction includes aproteolytic event comprising the foregoing steps.

The term “modulator” thus refers to a chemical compound (naturallyoccurring or synthesised), such as a biological macromolecule (e.g.,nucleic acid, protein, non-peptide, or organic molecule), or an extractmade from biological materials such as bacteria, plants, fungi, oranimal particularly mammalian) cells or tissues, or even an inorganicelement or molecule. Modulators are evaluated for potential activity asinhibitors or activators (directly or indirectly) of a biologicalprocess or processes (e.g., agonist, partial antagonist, partialagonist, antagonist, antineoplastic agents, cytotoxic agents, inhibitorsof neoplastic transformation or cell proliferation, cellproliferation-promoting agents, and the like) by inclusion in screeningassays described herein. The activities (or activity) of a modulator maybe known, unknown or partially-known. Such modulators can be screenedusing the methods described herein.

The term “candidate modulator” refers to a compound to be tested by oneor more screening method(s) of the invention as a putative modulator.Usually, various predetermined concentrations are used for screeningsuch as 0.01 μM, 0.1 μM, 1.0 μM, and 10.0 μM, as described more fullybelow. Test compound controls can include the measurement of a signal inthe absence of the test compound or comparison to a compound known tomodulate the target.

“Modulation” refers to the capacity to either increase or decease theproteolytic activity of a protease enzyme by at least 10%, 15%, 20%,25%, 50%, 100% or more; such increase or decrease may be contingent onthe occurrence of a specific event, such as activation of a signaltransduction pathway, and/or may be manifest only in particular celltypes.

In a still further aspect, the invention provides the use of apolypeptide multimer according to the preceding aspects of the inventionfor the detection or monitoring of a protease activity.

Other features and advantages of the invention ae found in the detaileddescription of the invention, and in the following claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of a homo-oligomeric assay ofthe invention in which each of the polypeptides comprises a proteasecleavage site.

FIG. 2 is a diagrammatic representation of a heteromultimeric assay ofthe invention in which one or more of the polypeptides comprises aprotease site.

FIG. 3 is a diagrammatic representation of a multimeric assay of theinvention in which one of the polypeptides is produced as a fusionprotein with an additional polypeptide, and assayed for proteolyticdegradation.

FIG. 4 is a diagrammatic representation of a single-polypeptide reagentuseful according to the invention.

FIG. 5 is a diagrammatic representation of a single-polypeptide reagentuseful according to the invention.

FIG. 6 depicts constructs useful according to the invention.

FIG. 7 depicts a reporter molecule useful according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

Generally, the nomenclature used herein and the laboratory procedures incell culture, molecular genetics, and nucleic acid chemistry andhybridisation described below are those well known and commonly employedin the art. Standard techniques are used for recombinant nucleic acidmethods, polynucleotide synthesis, and microbial culture andtransformation (e.g., electroporation, lipofection). Generally,enzymatic reactions and purification steps are performed according tothe manufacturer's specifications. The techniques and procedures aregenerally performed according to conventional methods in the art andvarious general references (see generally, Sambrook et al., MolecularCloning: A Laboratory Manual 2d ed. (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., which is incorporated herein byreference) which are provided throughout this document. The nomenclatureused herein and the laboratory procedures in analytical chemistry,organic synthetic chemistry, and pharmaceutical formulation describedherein are those well known and commonly employed in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical formulation and delivery, and treatment of patients.

The present invention may be configured in a number of ways. Exemplaryembodiments of the invention are set forth below.

Design and Construction of Polypeptides

Polypeptides useful in the present invention are capable ofmultimerising, either with similar or different polypeptides, to form apolypeptide multimer in accordance with the invention. Such polypeptidesmay be naturally-occurring polypeptides, modified naturally-occurringpolypeptides, or artificial polypeptides. Naturally-occurringpolypeptides may be isolated from natural sources or, preferably,synthesised by peptide synthesis or produced using recombinant DNAexpression technology. Synthetic or partially synthetic polypeptides maybe synthesised by peptide synthesis or using recombinant DNA technologyand, if necessary, nucleic acid synthesis techniques. Various techniquesfor the synthesis of nucleic acids and peptides are known in the art andmay be applied to the present invention.

In a preferred embodiment, labelled polypeptides may be produced via theexpression of recombinant nucleic acid molecules comprising an in-framefusion of sequences encoding a desired polypeptide and a fluorescentprotein moiety either in vitro (e.g., using a cell-freetranscription/translation system, as described below, or instead usingcultured cells transformed or transfected using methods well known inthe art) or in vivo, for example in a transgenic animal including, butnot limited to, insects, amphibians and mammals. A recombinant nucleicacid molecule of use in the invention may be constructed and expressedby molecular methods well known in the art, and may additionallycomprise sequences including, but not limited to, those which encode atag (e.g., a histidine tag) to enable easy purification, a secretionsignal, a nuclear localisation signal or other primary sequence signalcapable of targeting the construct to a particular cellular location, ifit is so desired.

The use of heteromultimers is advantageous in several ways —unproductivelabel partnerships are outlawed; assays can be configured such that anumber of modifications can be monitored using unique, modifiedpolypeptides peculiar to each assay and one common polypeptide, which isimportant in the automation of assays; moreover, where only onepolypeptide is susceptible to cleavage, only a single cleavage event ispossible per multimer, which increases the output of the assay asunproductive cleavages are avoided.

The structural requirements for a polypeptide useful in the presentinvention may be defined as follows. Firstly, the polypeptide requires abinding site which will permit it to bind to other polypeptides to forma multimer. Polypeptides are known to be able to associate in a numberof ways, and domains which mediate polypeptide association are alsoknown. For example, the coiled coil domain is known to mediateprotein—protein interactions, as are variants including both canonicaland non-canonical coiled-coil repeat structures, such as leucine zipperdomains, and non-coiled-coil domains such as SH2 domains and SH3domains.

Table 5 sets forth a number of protein binding domains useful in thepresent invention.

TABLE 5 Example Possible positions positions for for Sub- Partnerengineered Partner engineered Class class 1 sites 2 sites INTRA- PKC PKCMOLECU- pseudo- RACK LAR RACK binding site^(□) site^(□) Hemolin Hemolindomains domains 1, 2 3, 4 M63398 M63398 HOMO- PKA 1-36 PKA 1-36 OLIGOMERRIIβ RIIβ M31158 M31158 MetJ 20-29, MetJ 20-29, monomer 52-66 monomer52-66 M12869 M12869 Phospho- 18-31 Phospho- 18-31 lamban lamban M60411M60411 HETERO- SH2‡ Src 150-247 RACK1 OLIGOMER K03218 M24194 Src 147-244AFAP110 J00844 L20303 RasGAP 181-272, EphB2 juxta- M23379 351-441 L25890membrane (human) (mouse) region, AF025304 including (human) 604-613(mouse) SH3 ArgBP2 436-484, Arg** pro AF049884 614-664 rich region 2CRKL 123-296 Abl 1 782-1019 X59656 X16416 PDZ nNOS 1-195 PSD 95 138-294U17327 U83192 PTP-BL 1352- RIL 249-330 Z32740 1450, Y08361 (LIM 1756-domain) 1855 PH RAC 1-108 PKC 1-250 protein ζ_* kinase β_(—) M77198 βARK556-670 Gβγ_{overscore (ω)} WD40 X61157 repeats 5 and 6 PTB φ IRS 1157-267 ILA-R 489-499 S62539 X52425 Cbl 1-357 ZAP 70 284-299 X57110L05148 WW Nedd4 218-251, Amilo- C- D42055 375-408, ride- terminal448-481, sensitive P2 500-533 Na+ region channel β subunit L36593 γsubunit L36592 AKAP AKAP 79 388-409 PKA 1-36 M90359 RIIβ M31158 AKAP 7931-52 PKC α, M90359 β1, β2 ζ AKAP 79 81-102 Calcineu- M90359 rin (Asubunit) M81483 Gravin 1537- PKA 1-45 U81607 1563 RIIβ M31158 Gravin265-556 PKC β2^(Å) U81607 RACK RACK1 PKCβ1 186-198, M24194 X06318,209-226 M27545 b′COP PKCe 2-145 X70476 X65293, S46030 YXDED ZIP^(˜)41-105 PKC 79-145 Y08355 ζ^(˜) ^(□)Ron, D., Mochly-Rosen, D.,Proceedings of the National Academy of Sciences USA 1995, 92 492-496.‡The SH2 domain is modified such that the addition or removal of aphosphate group from a tyrosine residue is no longer a determinant ofbinding. This is achieved by thiophosphorylation of the Tyr residue inan in vitro assay to yield a permanently phosphorylated protein.Alternatively, it is possible to mimic phosphorylation by the mutationof the key Tyr residue to Glu or Asp. **Wang, B., Golemis, E. A., KruhG. D., Journal of Biological Chemistry 1997, 272 17542-17550^(ν)Konishi, H., Kuroda, S., Kikkawa, U., Biochemical and BiophysicalResearch Communications 1994, 205 1770-1775. {overscore (ω)}Fushman, D.,Najmabadi-Haske, T., Cahill, S., Zheng, J., LeVine III, H., Cowburn, D.,Journal of Biological Chemistry 1998, 273 2835-2843. φAgain, the PTBdomain is modified such that the addition or removal of a phosphategroup from a tyrosine residue is no longer a determinant of binding.This is achieved by thiophosphorylation of the Tyr residue in an invitro assay to yield a permanently phosphorylated protein.Alternatively, it is possible to mimic phosphorylation by the mutationof the key Tyr residue to Glu or Asp. ζKlauck, T. M., Faux, M. C.,Labudda, K., Langeberg, L. K., Jaken, S., Scott, J. D., Science 1996,271 1589-92 ^(Å)Nauert, J. B., Klauck, T. M., Langeberg, L. K., Scott,J. D., Current Biology 1997, 7 52-62 ^(˜)Puls, A., Schmidt, S., Grawe,F., Stabel, S., Proceedings of the National Academy of Sciences USA1997, 94 6191-6196.

ZIP contains more than one protein binding motif (YXDED motif, ZZ zincfinger) and is known to bind to several proteins other than PKC ζ(including_p62 and EBIAP) and also to self-associate (this selfassociation is in competition with PKC ζ binding). These multipleinteractions should be considered when designing an assay according tothe present invention.

The coiled-coil domain is structurally conserved among many proteinsthat interact to form homo- or heterodimeric oligomers. The leucinezipper provides an example of one such protein transcriptional activatorof a family of genes involved in the General Control of Nitrogen (GCN4)metabolism in S. cerevisiae. The protein is able to dimerise and bindpromoter sequences containing the recognition sequence for GCN4, therebyactivating transcription in times of nitrogen deprivation.

Coiled-coils are α-helical oligomers or bundles with between 1 and 5polypeptide strands with the following characteristics: (i) a sequencehallmark of a predominance of hydrophobic residues (in particularalanine, isoleucine, leucine, methionine or valine) spaced 3 and 4residues apart in the primary sequence which is repeated three or moretimes in near or exact succession (canonical heptad coiled-coil repeat,abbreviated to (3, 4)_(n), where n=3 or greater). The hydrophobicresidues are present at the ‘a’ and ‘d’ positions within a heptad whenthe amino acids are identified as positions a, b, c, d, e, f and g byorder of sequence. In addition, spacing of hydrophobic residues inpatterns of 3, 4, 4 and 3, 4, 3 (hendecad repeat) have recently beenreported (Hicks et al., 1997, Folding and Design, 2: 149-158) and arecompatible with the coiled-coil structure. (ii) In structural termscoiled-coil helical bundles have between 2 and 5 helices which areoffset at roughly 20° to adjacent strands with the hydrophobicsidechains interdigitating in the interface between helices in what istermed the “knobs into holes” packing (Crick, 1953, Acta Crystallogr, 6:689-697). Natural and non-natural coiled-coils can have parallel and/orantiparallel helices. Both homotypic (multiple strands of identicalsequence) and heterotypic bundles have been described.

Leucine zipper sequences conform to the coiled-coil rules above andtypically have leucine residues at the ‘d’ position of the canonicalheptad repeat. These leucine residues represent a single face of thehelix. Interdigitating with these leucine residues are other hydrophobicamino acids, frequently valine, isoleucine or leucine residues. Thecombination of these residues forms a continuous hydrophobic face whichassociates with an equivalent region in an associating subunit.Alternatively the hydrophobic face can be discontinuous due tointerruptions in the heptad repeat sequence. This, however, does notinterfere with the ability of these coiled-coils to interact. Thestability of the dimer thus formed is conferred by the hydrophobicinteractions between the leucine residues and the interdigitatinghydrophobic residues. Hydrogen bonds that form between residues presenton the two interacting helices, particularly at the e and g positions,also contribute to the stability of the dimer. The coiled-coil domain ofGCN4 has been shown to dimerise as an isolated peptide (Gonzalez et al.,1996, Nature Structural Biology, 3: 1011-1018).

Examples of naturally-occurring coiled-coils are as follows:

Coiled-oil class and example:

fgabcdefgabcdefgabcdefgabcdefgabcdefgabcdeg Parallel two-strandedtropomyosin TMPA_RABIT, 10-279 (270) (J. BIOL. CHEM. 253, 1137-1148,1978) dystrophin ILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHK SWISSPROT:P11532 (HUMAN) (SEQ ID NO:3) (Trends Biol. Sci., 20, 133-135, 1995)GCN4* MKQLEDKVEELLSKNYHLENEVARLKKLVGER GCN4_YEAST, 250-281 (32) (SEQ IDNO:4) (Proc. Natl. Acad. Sci. U.S.A., 81, 6442-6446, 1982) cFOS*TDTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAH FOS_HUMAN, 162-199 (39) (SEQ IDNO:5) (Proc. Natl. Acad. Sci. U.S.A., 80: 3183-3187, 198) cJUN*IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNH AP1_HUMAN, 277-315 (39) (SEQ IDNO:6) (Proc. Natl. Acad. Sci. U.S.A., 85: 9148-9152, 1988 antiparalleltwo-stranded Seryl-tRNA synthetase, E. coli*VDKLGALEERRKVLQVKTENLQAERNSRSKSIGQAKAR SYS_ECOLI, 27-64 (38) (SEQ IDNO:7) NUCLEIC ACIDS RES., 15, EPLRLEVNKLGEELDAAKAELDALQAEIRDIA1005-1017, 1987 (SEQ ID NO:8) SYS_ECOLI, 69-100 (32) Seryl-tRNAsynthetase, DLEALLALDREVQELKKRLQEVQTERNQVAKRV Thermus thermophilus* (SEQID NO:9) SYS_THERM, 26-58 (33) EALIARGKALGEEAKRLEEALREKEARLEALL(Science, 263: 1404-141) (SEQ ID NO:10) SYS_THERM, 67-98 (32) Transcriptcleavage factor GreA* LRGAEKLREELDFLKSvFRPEIIAAIAEAR GREA_ECOLI, 8-37(30) (SEQ ID NO:11) (Nature, 373: 636-640, 1995)AEYHAAREQQGFCEGRIKDIEAKLSN GREA_ECOLI, 46-71 (26) (SEQ ID NO:12)Parallel three-stranded GCN4 Zip mutant pll*MKQIEDKIEEILSKIYHIENEIARIKKLIGER GCN4 Zip mutant pll* (SEQ ID NO:13)(Nature, 371: 80-83) Antiparallel three-stranded synthetic peptidecoil-Ser* EWEALEKKLAALESKLQALEKKLEALEHG (Science, 259: 1288-1293) (SEQID NO:14) Parallel four-stranded GCN4 Zip mutant pLl*MKQIEDKLEEILSKLYHIENELARIKKLLGER (Nature, 371: 80-83) (SEQ ID NO:15)Antiparallel four-stranded Repressor of primer ROP*QEKTALNMARFIRSQILTLLEKLNE ROP_ECOLI, 4-28 (25) (SEQ ID NO:16) (Proc.Natl. Acad. Sci. U.S.A., DEQADICESLHDHADELYRSCLAR 79: 6313-6317 1982)(SEQ ID NO:17) ROP_ECOLI, 32-55 (24) Parallel five-strandedphospholamban LILICLLLICIIVMLL PPLA_HUMAN, 37-52 (16) (SEQ ID NO:18)(JBC 271, 5941-5946, 1996)

The binding domains may be similar or different, i.e. homomultimeric orheteromultimeric. The rules for the design of hetero-multimeric coiledcoils are well detailed in the literature (including Peptide ‘Velcro’:design of a heterodimeric coiled coil, O'Shea, E. K., Lumb, K. J. & Kim,P. S., Current Biology (1993) 3 658-667; A designed heterotrimericcoiled coil, Nautiyal, S., Woolfson, D. N., King, D. S. & Alber T.,Biochemistry (1995) 34 11645-11651; A buried polar interaction impartsstructural uniqueness in a designed heterodimeric coiled coil, Lumb, K.J. & Kim P. S., Biochemistry (1995) 34 8642-8648). The use of a‘designer’ coiled coil has benefit in providing control over thepotential modification sites present other than those inserted tomonitor the reaction of interest.

Of course, polypeptides may also associate via interactions, notnecessarily involving canonical domains such as coiled-coils, which maybe specific to the polypeptides in question.

Secondly, one or more polypeptides in each multimer may comprise alabel. Suitable fluorescent labels include fluorophores and fluorescentproteins. As used herein, the terms “fluorophore” and “fluorochrome”refer interchangeably to a molecule which is capable of absorbing energyat a wavelength range and releasing energy at a wavelength range otherthan the absorbance range. The term “excitation wavelength” refers tothe range of wavelengths at which a fluorophore absorbs energy. The term“emission wavelength” refers to the range of wavelength that thefluorophore releases energy or fluoresces.

A non-limiting list of chemical fluorophores of use in the invention,along with their excitation and emission wavelengths, is presented inTable 1.

TABLE 1 Excitation Emission Fluorophore (nm) (nm) Colour PKH2 490 504green PKH67 490 502 green Fluorescein (FITC) 495 525 green Hoechst 33258360 470 blue R-Phycoerythrin (PE) 488 578 orange-red Rhodamine (TRITC)552 570 red Quantum RedÔ 488 670 red PKH26 551 567 red TEXAS RED ™ 596620 red Cy3 552 570 red (Indodicarbocyanine-3)

Examples of fluorescent proteins which vary among themselves inexcitation and emission maxima are listed in Table 1 of WO 97/28261(incorporated herein by reference). These (each followed by [excitationmax./emission max.] wavelengths expressed in nanometers) includewild-type Green Fluorescent Protein [395(475)/508] and the cloned mutantof Green Fluorescent Protein variants P4 [383/447], P4-3 [381/445], W7[433(453)/475(501)], W2 [432(453)/480], S65T [489/511], P4-1[504(396)/480], S65A [471/504)]S65C [479/507], S65L [484/510], Y66F[360/442], Y66W [458/480], I0c [513/527], WIB [432(453)/476(503)],Emerald [487/508] and Sapphire [395/511]. This list is not exhaustive offluorescent proteins known in the art; additional examples are found inthe Genbank and SwissProt public databases.

A number of parameters of fluorescence output are envisaged including

-   -   1) measuring fluorescence emitted at the emission wavelength of        the acceptor (A) and donor (D) and determining the extent of        energy transfer by the ratio of their emission amplitudes;    -   2) measuring the fluorescence lifetime of D;    -   3) measuring the rate of photobleaching of D;    -   4) measuring the anistropy of D and/or A; or    -   5) measuring the Stokes shift monomer:eximer fluorescence.

Other labels may be used, however, depending on the detection methodemployed to monitor the signal generated by the label. Labels may beattached in a number of ways, such as by direct labelling at suitableamino acids, such as cysteines or lysines, with chemical labels, or byfusion with a polypeptide-label such as a fluorescent polypeptide.Techniques for labelling polypeptides are generally known in the art andmay be applied to the present invention.

The invention may be configured to exploit a number of non-fluorescentlabels. In a first embodiment, the polypeptide multimer is an enzymewhich is capable of participating in an enzyme-substrate reaction whichhas a detectable endpoint. The enzyme may be cleaved into two or morecomponents, such that upon multimer formation the components reassembleto form a functional enzyme. Enzyme function may be assessed by a numberof methods, including scintillation and photospectroscopy.

Cleavage of one or more polypeptides according to the invention isrequired to preclude multimer formation and enzyme component assembly,thus reducing enzyme activity.

In a second embodiment, an enzyme is used together with a modulator ofenzyme activity, such as an inhibitor or a cofactor. Binding of theenzyme and its inhibitor or cofactor results in modulation of enzymaticactivity, which is detectable by conventional means.

In a third embodiment, the invention is configured as a two-hybrid assay(Fields & Song, (1989), Nature 340, 245-6), in which two components of atranscription factor are used as polypeptides according to theinvention. Assembly of the transcription factor results in activation ofa transcription unit, with a resultant biological signal; a preferredbiological signal is luciferase expression, which is easily assessed.Cleavage of one or more of the components results in downregulation ofthe transcription unit and thus loss of signal.

In any of the foregoing embodiments, assembly of the enzyme ortranscription factor components may be spontaneous, such that the enzymeor transcription factor components are themselves the polypeptidesaccording to the invention. Furthermore, it may be dependent upon theassociation of associated binding domains, such that the enzyme andtranscription factor components are effectively labels according to thepresent invention. A two-hybrid type of assay is preferably configuredin this manner.

Thirdly, one or more of the polypeptides in each multimer according tothe invention must be susceptible to digestion by a protease enzyme. Asnoted above, susceptibility to digestion indicates that the polypeptidemay be subjected to proteolytic degradation under the appropriateconditions, which in a preferred embodiment means that the polypeptideis cleaved by a protease at a recognition site for the protease enzyme.Alternatively, however, the polypeptide may be susceptible to digestionby an exoprotease, from the N or C terminus. Peptides may be renderedsusceptible to protease digestion by inclusion within the peptidesequence of a recognition site for a protease. This may be performedusing peptide synthesis techniques as described above.

Polypeptides according to the invention should be constructed such thatthe protease cleavable site is positioned such that cleavage thereofdisrupts binding of the polypeptide in the context of the multimer.Thus, polypeptides which have been subjected to protease cleavage shoulddissociate from the multimer. Preferably, the protease does not cleavethen polypeptide in such a manner that the label becomes detachedtherefrom without the binding abilities thereof being disrupted.Location of the protease cleavable site may be determined empirically.As a guide, however, the site should be placed within or proximal to thebinding domain which is responsible for the multimerisation of thepolypeptide.

In the case of coiled coil binding domains, Lumb et al (Subdomainfolding of the coiled coil leucine zipper from the bZIP transcriptionalactivator GCN4, Lumb, K. J., Carr, C. M. & Kim, P. S., Biochemistry(1994) 33 7361-7367) teach that the loss of ten residues from theN-terminus or seven from the N- and six from the C-terminus issufficient to destabilise the coiled coil peptide sequence known asGCN4-p1 (Evidence that the leucine zipper is a coiled coil, O'Shea, E.K., Rutkiowski, R. & Kim, P. S. (1989) Science 243 538-542). Su et alprovide data indicating that there is a sharp decrease in the stabilityof a designed coiled coil with a decrease in chain length from 23 to 19residues (Su, J. Y., Hodges, R. S. & Kay, C. M., Biochemistry (1994) 3315501-15510). Accordingly, cleavage sites may positioned such that thecoiled coil is disrupted to an extent that it is no longer capable ofdirecting multimerisation.

The cleavage sites of a number of proteases are known in the art, andset forth in Table 2.

TABLE 2 Protease Cut Site(s) Possible/Proven Role Aminopeptidase MHydrolysis from digestion free N-terminus Carboxypeptidase P Hydrolysisfrom digestion C-terminus Carboxypeptidase Y Hydrolysis from digestionC-terminus Caspase 1, 4, 5 W/LEHD-X^(#) mediator of (SEQ ID NO:19)apoptosis Caspase 2, 3, 7 DEXD-X^(#) mediator of (SEQ ID NO:20)apoptosis Caspase 6, 8, 9 L/VEXD-X^(#) mediator of (SEQ ID NO:21)apoptosis Chymotrypsin Y-X, F-X, T-X, digestion (L-X, M-X, A-X, E-X)Factor Xa IEGR-X blood clotting (SEQ ID NO:22) cascade Pepsin F-Z, M-Z,L-Z, W-Z digestion (where Z is a hydrophobic residue) but will cleaveothers TEV E(N)XYXQ-S/G^(˜) polyprotein (SEQ ID NO:23) processing/as areagent Thrombin R-X blood clotting cascade Trypsin R-X, K-X digestion^(#)Ideal cut sites identified by Thornberry et al in A combinatorialapproach defines specificities of members of the caspase family andgranzyme B, Journal of Biological Chemistry 272 17907-17911. ^(˜)Releaseof proteins and peptides from fusion proteins using a recombinant plantvirus proteinase, Parks, T. D., Keuther, K. K., Howard, E. D., Johnston,S. A. & Dougherty, W. G., Analytical Biochemistry (1994) 216 413-417;Life Technologies Ltd.

The foregoing, or other, sites may be engineered into or close to thebinding domains of polypeptides according to the invention.

In a preferred aspect of the present invention, it is desirable toengineer specificity into the polypeptide, such that it is digested onlyby the desired protease and only at the intended protease cleavablesite. This may be achieved, for example, by the use of D-isomers ofamino acids in the construction of the polypeptide. D-amino acids areresistant to protease digestion, and a polypeptide constructed ofD-amino acids will withstand proteolytic attack. Moreover, use ofD-amino acids does not interfere with the protein—protein interactionsinvolved in multimerisation, such as the interaction of protein bindingdomains, especially coiled-coil domains, provided that D amino acids areemployed in both members of a binding pair.

In order to allow digestion by the intended protease enzyme, the D-aminoacid constructions of the polypeptides of the invention contain one ormore parts constructed of L-amino acids, or otherwise renderedsusceptible to proteolytic digestion. For example, coiled coilsconstructed of D-amino acids preferably comprise inserts, constructedwholly or partly of L-amino acids, which contain the protease cleavagesite. The L-amino acid insert may be of any size, and may be positionedbetween coiled coil repeats, or between residues of the coiled coil.Preferred are insertions between residues b-c, e-f and f-g. The insertis covalently attached to the coiled coil, through a peptide linkage tothe backbone or through a sidechain.

The insert may comprise only a cleavage site, or an entire polypeptide.Functionally, the insert is sufficiently flexible to permit the coiledcoil to bind to its target efficiently when the insert is intact. Forexample, the insert may comprise a flexible linker, such as a gly—glylinker. Molecules comprising D-amino acids are advantageously employedin in vitro assays.

Inserts as described above may be employed in D-amino acid coiled coils,in conventional L-amino acid coiled coils, or in coiled coils which arepartially D and partially L in construction. Foe example, a coiled coilmay be constructed such that it consist of L-amino acids on one side ofthe insert, and D-amino acids on the other side thereof.

Generation of a Detectable Signal

Depending on the embodiment in question, a signal useful in the presentinvention may be generated by a number of different labels. Preferredare fluorescent labels, and particularly preferred are fluorescentlabels which participate in energy transfer (FRET).

FRET is detectable when two fluorescent labels which fluoresce atdifferent frequencies are sufficiently close to each other that energyis able to be transferred from one label to the other. FRET is widelyknown in the art (for a review, see Matyus, 1992, J. Photochem.Photobiol. B: Biol., 12: 323-337, which is herein incorporated byreference). FRET is a radiationless process in which energy istransferred from an excited donor molecule to an acceptor molecule; theefficiency of this transfer is dependent upon the distance between thedonor and acceptor molecules, as described below. Since the rate ofenergy transfer is inversely proportional to the sixth power of thedistance between the donor and acceptor; the energy transfer efficiencyis extremely sensitive to distance changes. Energy transfer is said tooccur with detectable efficiency in the 1-10 nm distance range, but istypically 4-6 mm for favourable pairs of donor and acceptor.

Radiationless energy transfer is based on the biophysical properties offluorophores. These principles are reviewed elsewhere (Lakowicz, 1983,Principles of Fluorescence Spectroscopy, Plenum Press, New York; Jovinand Jovin, 1989, Cell Structure and Function by Microspectrofluorometry,eds. E. Kohen and J. G. Hirschberg, Academic Press, both of which areincorporated herein by reference). Briefly, a fluorophore absorbs lightenergy at a characteristic wavelength. This wavelength is also known asthe excitation wavelength. The energy absorbed by a fluorochrome issubsequently released through various pathways, one being emission ofphotons to produce fluorescence. The wavelength of light being emittedis known as the emission wavelength and is an inherent characteristic ofa particular fluorophore. Radiationless energy transfer is thequantum-mechanical process by which the energy of the excited state ofone fluorophore is transferred without actual photon emission to asecond fluorophore. That energy may then be subsequently released at theemission wavelength of the second fluorophore. The first fluorophore isgenerally termed the donor (D) and has an excited state of higher energythan that of the second fluorophore, termed the acceptor (A). Theessential features of the process are that the emission spectrum of thedonor overlap with the excitation spectrum of the acceptor, and that thedonor and acceptor be sufficiently close. The distance over whichradiationless energy transfer is effective depends on many factorsincluding the fluorescence quantum efficiency of the donor, theextinction coefficient of the acceptor, the degree of overlap of theirrespective spectra, the refractive index of the medium, and the relativeorientation of the transition moments of the two fluorophores. Inaddition to having an optimum emission range overlapping the excitationwavelength of the other fluorophore, the distance between D and A mustbe sufficiently small to allow the radiationless transfer of energybetween the fluorophores.

FRET may be performed either in vivo or in vitro. Proteins are labelledeither in vivo or in vitro by methods known in the art. According to theinvention, two coiled-coil domains comprised either by the same or bydifferent polypeptide molecules are differentially labelled, one with adonor and the other with an acceptor moiety, and differences influorescence between a test assay, comprising a protein modifyingenzyme, and a control, in which the modifying enzyme is absent, aremeasured using a fluorimeter or laser-scanning microscope. It will beapparent to those skilled in the art that excitation/detection means canbe augmented by the incorporation of photomultiplier means to enhancedetection sensitivity. The differential labels may comprise either twodifferent fluorescent moieties (e.g., fluorescent proteins as describedbelow or the fluorophores rhodamine, fluorescein, SPQ, and others as areknown in the art) or a fluorescent moiety and a molecule known to quenchits signal.

In a FRET assay of the invention, the fluorescent labels are chosen suchthat the excitation spectrum of one of the labels (the acceptor label)overlaps with the emission spectrum of the excited fluorescent label(the donor label). The donor label is excited by light of appropriateintensity within the donor's excitation spectrum. The donor then emitssome of the absorbed energy as fluorescent light and dissipates some ofthe energy by FRET to the acceptor fluorescent label. The fluorescentenergy it produces is quenched by the acceptor fluorescent label. FRETcan be manifested as a reduction in the intensity of the fluorescentsignal from the donor, reduction in the lifetime of its excited state,and re-emission of fluorescent light at the longer wavelengths (lowerenergies) characteristic of the acceptor. When the donor and acceptorlabels become spatially separated, FRET is diminished or eliminated.

One can take advantage of the FRET exhibited by two polypeptideslabelled with different fluorescent labels, wherein one polypeptide islinked to a donor and another to an acceptor label, in monitoringproteolytic digestion according to the present invention. Two distinctpolypeptides each comprising a coiled-coil may be differentiallylabelled with the donor and acceptor fluorescent protein moieties,respectively.

The means by which polypeptides are assayed for association usingfluorescent protein moiety labels according to the invention may bebriefly summarised as follows:

-   -   Of two polypeptides which associate into a multimer according to        the present invention, one is labelled with a green fluorescent        protein, while the other is preferably labelled with a red or,        alternatively, a blue fluorescent protein. Useful donor:acceptor        pairs of fluorescent proteins (see WO 97/28261) include, but are        not limited to:

-   Donor: S72A, K79R, Y145F, M153A and T2031 (excitation 395 nm;    emission 511)

-   Acceptor: S659, S72A, K79R and T203Y (wavelengths not noted), or    T203Y/S65G, V68L, Q69K or S72A (excitation 515 nm; emission 527 nm).

An example of a blue:green pairing is P4-3 (shown in Table 1 of WO97/28261) as the donor moiety and S65C (also of Table 1 of WO 97/28261)as the acceptor moiety. The polypeptides comprising coiled-coils areexposed to light at, for example, 368 nm, a wavelength that is near theexcitation maximum of P4-3. This wavelength excites S65C only minimally.Upon excitation, some portion of the energy absorbed by the bluefluorescent protein moiety is transferred to the acceptor moiety throughFRET if the two polypeptides comprising coiled-coils are in closeassociation. As a result of this quenching, the blue fluorescent lightemitted by the blue fluorescent protein is less bright than would beexpected if the blue fluorescent protein existed in isolation. Theacceptor moiety (S65C) may re-emit the energy at longer wavelength, inthis case, green fluorescent light.

After proteolytic degradation, the polypeptides physically separate,accordingly inhibiting FRET. Such a system is useful to monitor theactivity of proteolytic enzymes that cleave polypeptides to which thefluorescent labels are fused as well as the activity of modulators orcandidate modulators of those enzymes.

In particular, the invention contemplates assays in which the amount oractivity of a protease in a sample is determined by contacting thesample with a pair of polypeptides differentially labelled withfluorescent proteins, as described above, and measuring changes influorescence of the donor moiety, the acceptor moiety or the relativefluorescence of both. Fusion proteins, as described above, can be usedfor, among other things, monitoring the activity of a protease enzymeinside the cell that expresses two different recombinant constructs.

Advantages of fluorescent polypeptides constructed as fusions withfluorescent proteins include the greater extinction coefficient andquantum yield of many of these proteins compared with those of the Edansfluorophore. Also, the acceptor in such a construct or pair ofconstructs is, itself, a fluorophore rather than a non-fluorescentquencher like Dabcyl. Thus, the enzyme's substrate, i.e., the unmodifiedpolypeptide of the construct(s) and products (i.e., the polypeptidesafter digestion) are both fluorescent but with different fluorescentcharacteristics.

In particular, the substrate and modified products exhibit differentratios between the amount of light emitted by the donor and acceptorlabels. Therefore, the ratio between the two fluorescences measures thedegree of conversion of substrate to products, independent of theabsolute amount of either, the optical thickness of the sample, thebrightness of the excitation lamp, the sensitivity of the detector, etc.Furthermore, Aequorea-derived or related fluorescent protein moietiestend to be protease resistant. Therefore, they are likely to retaintheir fluorescent properties throughout the course of an experiment.

Additional embodiments of the present invention are not dependent onFRET. For example the invention can make use of fluorescence correlationspectroscopy (FCS), which relies on the measurement of the rate ofdiffusion of a label (see Elson & Magde, (1974) Biopolymers 13:1-27;Rigler et al., (1992) in Fluorescence Spectroscopy New Methods andApplications, Springer Verlag, pp.13-24; Eigen & Rigler, (1994) PNAS(ISA) 91:5740-5747; Kinjo & Rigler, (1995) NAR 23:1795-1799).

In FCS, a focused laser beam illuminates a very small volume ofsolution, of the order of 10⁻¹⁵l, which at any given point in timecontains only one molecule of the many under analysis. The diffusion ofsingle molecules through the illuminated volume, over time, results inbursts of fluorescent light as the labels of the molecules are excitedby the laser. Each individual burst, resulting from a single molecule,can be registered.

A labelled polypeptide will diffuse at a slower rate if it is large thanif it is small. Thus, multimerised polypeptides will display slowdiffusion rates, resulting in a lower number of fluorescent bursts inany given timeframe, whilst labelled polypeptides which are notmultimerised or which have dissociated from a multimer will diffuse morerapidly. Binding of polypeptides according to the invention can becalculated directly from the diffusion rates through the illuminatedvolume.

Where FCS is employed, rather than FRET, it is not necessary to labelmore than one polypeptide. Preferably, a single polypeptide member ofthe multimer is labelled. The labelled polypeptide dissociates from themultimer as a result of protease digestion, thus altering the FCSreading for the fluorescent label.

A further detection technique which may be employed in the method of thepresent invention is the measurement of time-dependent decay offluorescence anisotropy. This is described, for example, in Lacowicz(1983) Principles of Fluorescence Spectroscopy, Plenum Press, New York,incorporated herein by reference. See, for example, page 167.

Fluorescence anisotropy relies on the measurement of the rotation offluorescent groups. Larger multimers of polypeptides rotate more slowlythan monomers, allowing the formation of multimers to be monitored.

Protease Digestion

Digestion of polypeptides according to the invention with proteolyticenzymes may be carried out either in vivo or in vitro, according totechniques and procedures known in the art. Thus, appropriate amounts ofpolypeptides according to the invention are incubated in appropriateconditions, for example with an appropriate buffer.

As used herein, the term “appropriate buffer” refers to a medium whichpermits activity of the protease enzyme used in an assay of theinvention, and is typically a low-ionic-strength buffer or otherbiocompatible solution (e.g., water, containing one or more ofphysiological salt, such as simple saline, and/or a weak buffer, such asTris or phosphate, or others as described hereinbelow), a cell culturemedium, of which many are known in the art, or a whole or fractionatedcell lysate. An “appropriate buffer” permits digestion of polypeptidesaccording to the invention and, preferably, inhibits degradation andmaintains biological activity or the reaction components. Inhibitors ofdegradation, such as nuclease inhibitors (e.g., DEPC) are well known inthe art. Lastly, an appropriate buffer may comprise a stabilisingsubstance such as glycerol, sucrose or polyethylene glycol.

As used herein, the term “appropriate amounts of polypeptides” refers toan amount of labelled polypeptides of the invention which emit a signalwithin the detection limits of a measuring device used in an assay ofthe invention. Such an amount is great enough to permit detection of asignal, yet small enough that a change in signal emission is detectable(e.g., such that a signal is below the upper limit of the device).

Configuration of the Invention Using a Homomultimer Comprising aProtease Cleavage Site

In one embodiment of the present invention, the assay may be configuredto use a homomultimer of polypeptides, which are cleavable by a proteaseat a specific protease cleavable site.

This assay requires the formation of an oligomer of binding partnerscontaining a proteolytic site or with a proteolytic site engineeredtherein which are labelled with fluorophores appropriate for FRET (orsome other appropriate means of detection). The coiled coil provides agood example of such an oligomer. The sequence of the coiled coil partof the peptides in this reporter is advantageously short, such that itcontains at least two heptad structures and preferably about 4 or 5heptads. Within this heptad structure an amino acid sequence recognisedas a cleavage site for a protease is included. Cleavage of the peptideby the protease will reduce the length of the continuous coiled coilsequence, and destabilise oligomer formation. The fluorophores (F1, F2;where F1 is the donor fluorophore and F2 the acceptor) should bepositioned such that they are covalently attached to separate peptidesin the oligomeric complex at positions which are close in space in thiscomplex but are not attached to a residue which prevents enzymeprocessing of the substrate.

An assay based on a homo-oligomeric assembly of peptides is representedin FIG. 1.

Configuration of the Invention using a Heteromultimer Comprising aProtease Cleavage Site

The assay may, however, also be configured as a heteromultimeric assayin which one or more of the polypeptides comprises a protease cleavagesite.

Clearly, the assays may be modified with respect to the diagram in FIG.2 in order to permit proteolytic degradation of more than onepolypeptide in the multimer. However, one of the advantages ofheteromultimeric assays is that only a single polypeptide is digested.

Configuration of the Invention Using Heteromultimer

Susceptible to Proteolytic Degradation

The invention may be configured to monitor the degradation ofpolypeptides, especially naturally-occurring polypeptides, as a resultof proteolytic degradation. For instance, a polypeptide may be producedas a fusion with a further polypeptide which is to be assayed forproteolytic degradation. Protease enzymes which degrade the furtherpolypeptide will also degrade the polypeptide of the invention, givingrise to multimer dissociation (FIG. 3).

Since the multimer is, in the embodiment, potentially much larger thanthe labelled dissociated monomer, the use of detection techniquesreliant on label diffusion such as FCS is facilitated. Moreover, the useof single labelled polypeptides, in conjunction with FCS detection, ismade possible.

Configuration of a Single-Polypeptide Binding Domains

Single-polypeptide reagents may be configured in a number of ways,depending on the location of the labels with respect to the bindingdomains on the polypeptide, In a first example (FIG. 4), the labels arepositioned N- and C-terminal to the respective binding domains.

Unfolded polypeptide molecule—no FRET.

Folded molecule—FRET between labels.

Cleavage of one binding domain leads to unfolding of the molecule andloss of FRET (FIG. 4).

In a second example one or more of the labels may not be cleaved fromthe molecule by the protease, but rely on distancing of the labels tolead to loss of FRET (FIG. 5).

Configuration Using Multiple Fluorescent Emissions

Interaction or association of polypeptides may be measured by anysuitable means as set out in this application. It will be recognised bythose skilled in the art that some means of detection allow themonitoring of multiple pairs of interacting polypeptides by choosing thefluorophore labels in such a way that different pairs of polypeptidesare labelled with different colour fluors (ie. FRET pairs which areconfigured such that their absorption and/or emission spectra aredifferent from one another).

By practising the invention in this manner, it will be possible tomeasure association of a first pair of polypeptides by exciting with afirst excitation wavelength and monitoring a first emission wavelengh,and to measure association of a second pair of polypeptides by excitingwith a second excitation wavelength and monitoring a second emissionwavelengh, both the first and second polypeptide pairs being present inthe same sample.

Clearly, two such pairs may have similar absorption wavelengths, withdifferent emission wavelengths, or may have different absorptionwavelengths with similar emission wavelengths, or may differ from oneanother in both their absorption and emission wavelengths. It will beunderstood that for the practice of the invention in this manner, theonly requirement is that the fluors are arranged in such a way that FRETpairs may be separately monitored or otherwise distinguished.

The only limitation to the number of polypeptide pairs which may bemonitored in solution in the same sample is the number of differentcombinations of suitable fluorophores which are available.

Fluorophores or FRET pairs with different absorption/emissionwavelengths are well known to those skilled in the art, and someexamples of these are presented in Table 1.

General Techniques Useful in the Invention

In the present invention, use is made of general techniques ofbiochemistry and molecular biology as described, for example, inSambrook et al. as referred to above. Generally, such techniques areuseful in the design of polypeptide molecules, and multimers thereof,according to the invention; the production thereof, especially byrecombinant DNA techniques; the attachment of labels to the molecules;in the incubation of molecules according to the invention with proteaseenzymes; and the design of assay protocols to monitor protease activity.

Design of Polypeptides

This is described in the foregoing sections on polypeptide design.

Production of Molecules

Molecules according to the invention are advantageously produced ininsect cell systems. Insect cells suitable for use in the method of theinvention include, in principle, any lepidopteran cell which is capableof being transformed with an expression vector and expressingheterologous proteins encoded thereby. In particular, use of the Sf celllines, such as the Spodoptera frugiperda cell line IPBL-SF-21 AE (Vaughnet al., (1977) In Vitro, 13, 213-217) is preferred. The derivative cellline Sf9 is particularly preferred. However, other cell lines, such asTricoplusia ni 368 (Kurstack and Marmorosch, (1976) Invertebrate TissueCulture Applications in Medicine, Biology and Agriculture. AcademicPress, New York, USA) may be employed. These cell lines, as well asother insect cell lines suitable for use in the invention, arecommercially available (e.g. from Stratagene, La Jolla, Calif., USA).

As well as expression in insect cells in culture, the invention alsocomprises the expression of polypeptides in whole insect organisms. Theuse of virus vectors such as baculovirus allows infection of entireinsects, which are in some ways easier to grow than cultured cells asthey have fewer requirements for special growth conditions. Largeinsects, such as silk moths, provide a high yield of heterologousprotein. The protein can be extracted from the insects according toconventional extraction techniques.

Expression vectors suitable for use in the invention include all vectorswhich are capable of expressing foreign proteins in insect cell lines.In general, vectors which are useful in mammalian and other eukaryoticcells are also applicable to insect cell culture. Baculovirus vectors,specifically intended for insect cell culture, are especially preferredand are widely obtainable commercially (e.g. from Invitrogen andClontech). Other virus vectors capable of infecting insect cells areknown, such as Sindbis virus (Hahn et al., (1992) PNAS (USA) 89,2679-2683). The baculovirus vector of choice (reviewed by Miller (1988)Ann. Rev. Microbiol. 42, 177-199) is Autographa californica multiplenuclear polyhedrosis virus, AcMNPV.

Typically, the heterologous gene replaces at least in part thepolyhedrin gene of AcMNPV, since polyhedrin is not required for virusproduction. In order to insert the heterologous gene, a transfer vectoris advantageously used. Transfer vectors are prepared in E. coli hostsand the DNA insert is then transferred to AcMNPV by a process ofhomologous recombination.

Alternatively, molecules according to the invention may be expressed inbacterial, lower eukaryote or mammalian cell systems, or in transgenicanimals.

cDNA or genomic DNA encoding polypeptides according to the invention canbe incorporated into vectors for expression. As used herein, vector (orplasmid) refers to discrete elements that are used to introduceheterologous DNA into cells for either expression or replicationthereof. Selection and use of such vehicles is well within the skill ofthe artisan. Many vectors are available, and selection of theappropriate vector will depend on the intended use of the vector, thesize of the DNA to be inserted into the vector, and the host cell to betransformed with the vector. Each vector contains various componentsdepending on its function and the host cell for which it is compatible.The vector components generally include, but are not limited to, one ormore of the following: an origin of replication, one or more markergenes, an enhancer element, a promoter, a transcription terminationsequence and a signal sequence.

Both expression and cloning vectors generally contain nucleic acidsequences that enable the vector to replicate in one or more selectedhost cells. Typically in cloning vectors, this sequence is one thatenables the vector to replicate independently of the host chromosomalDNA, and includes origins of replication or autonomously replicatingsequences. Such sequences are well known for a variety of bacteria,yeast and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2 m plasmid origin issuitable for yeast, and various viral origins (e.g. SV 40, polyoma,adenovirus) are useful for cloning vectors in mammalian cells.Generally, the origin of replication component is not needed formammalian expression vectors unless these are used in mammalian cellscompetent for high level DNA replication, such as COS cells.

Most expression vectors are shuttle vectors, i.e. they are capable ofreplication in at least one class of organisms but can be transfectedinto another class of organisms for expression. For example, a vector iscloned in E. coli and then the same vector is transfected into yeast ormammalian cells even though it is not capable of replicatingindependently of the host cell chromosome. DNA may also be replicated byinsertion into the host genome. DNA can be amplified by PCR and bedirectly transfected into the host cells without any replicationcomponent.

Advantageously, an expression and cloning vector may contain a selectiongene also referred to as selectable marker. This gene encodes a proteinnecessary for the survival or growth of transformed host cells grown ina selective culture medium. Host cells not transformed with the vectorcontaining the selection gene will not survive in the culture medium.Typical selection genes encode proteins that confer resistance toantibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate ortetracycline, complement auxotrophic deficiencies, or supply criticalnutrients not available from complex media.

As to a selective gene marker appropriate for yeast, any marker gene canbe used which facilitates the selection for transformants due to thephenotypic expression of the marker gene. Suitable markers for yeastare, for example, those conferring resistance to antibiotics G418,hygromycin or bleomycin, or provide for prototrophy in an auxotrophicyeast mutant, for example the URA3, LEU2, LYS2, TRP1, or HIS3 gene.

Since the replication of vectors is conveniently done in E. coli, an E.coli genetic marker and an E. coli origin of replication areadvantageously included. These can be obtained from E. coli plasmids,such as pBR322, Bluescript© (vector or a pUC plasmid, e.g. pUC18 orpUC19, which contain both E. coli replication origin and E. coli geneticmarker conferring resistance to antibiotics, such as ampicillin.

Suitable selectable markers for mammalian cells are those that enablethe identification of cells which have taken up vectors according to theinvention, such as dihydrofolate reductase (DHFR, methotrexateresistance), thymidine kinase, or genes conferring resistance to G418 orhygromycin. The mammalian cell transformants are placed under selectionpressure which only those transformants which have taken up and areexpressing the marker are uniquely adapted to survive. In the case of aDHFR or glutamine synthase (GS) marker, selection pressure can beimposed by culturing the transformants under conditions in which thepressure is progressively increased, thereby leading to amplification(at its chromosomal integration site) of both the selection gene and thelinked DNA that encodes a polypeptide according to the invention.Amplification is the process by which genes in greater demand for theproduction of a protein critical for growth, together with closelyassociated genes which may encode a desired protein, are reiterated intandem within the chromosomes of recombinant cells. Increased quantitiesof desired polypeptide are usually synthesised from thus amplified DNA.

Expression and cloning vectors usually contain a promoter that isrecognised by the host organism and is operably linked to a codingsequence. Such a promoter may be inducible or constitutive. Thepromoters are operably linked to DNA encoding a polypeptide according tothe invention by removing the promoter from the source DNA byrestriction enzyme digestion and inserting the isolated promotersequence into the vector. Both the native promoter sequence associatedwith the polypeptide in question, where this is naturally occurring, andmany heterologous promoters may be used to direct amplification and/orexpression of nucleic acids. The term “operably linked” refers to ajuxtaposition wherein the components described are in a relationshippermitting them to function in their intended manner. A control sequence“operably linked” to a coding sequence is ligated in such a way thatexpression of the coding sequence is achieved under conditionscompatible with the control sequences.

Promoters suitable for use with prokaryotic hosts include, for example,the β-lactamase and lactose promoter systems, alkaline phosphatase, thetryptophan (trp) promoter system and hybrid promoters such as the tacpromoter. Their nucleotide sequences have been published, therebyenabling the skilled worker to operably ligate them to DNA encoding apolypeptide according to the invention, using linkers or adaptors tosupply any required restriction sites. Promoters for use in bacterialsystems will also generally contain a Shine-Delgarno sequence operablylinked to the DNA encoding a polypeptide according to the invention.

Preferred expression vectors are bacterial expression vectors whichcomprise a promoter of a bacteriophage such as phagex or T7 which iscapable of functioning in the bacteria. In one of the most widely usedexpression systems, the nucleic acid encoding the fusion protein may betranscribed from the vector by T7 RNA polymerase (Studier et al, Methodsin Enzymol. 185; 60-89, 1990). In the E. coli BL21(DE3) host strain usedin conjunction with pET vectors, the T7 RNA polymerase is produced fromthe λ-lysogen DE3 in the host bacterium and its expression is under thecontrol of the IPTG inducible lac UV5 promoter. This system has beenemployed successfully for over-production of many proteins,Alternatively the polymerase gene may be introduced on a lambda phage byinfection with an int-phage such as the CE6 phage which is commerciallyavailable (Novagen, Madison, USA). Other vectors include vectorscontaining the lambda PL promoter such as PLEX (Invitrogen, NL), vectorscontaining the trc promoters such as pTrcHisXpress™ (Invitrogen) orpTrc99 (Pharmacia Biotech, SE), or vectors containing the tac promotersuch as pKK223-3 (Pharmacia Biotech) or PMAL (new England Biolabs, MA,USA).

Moreover, the nucleic acids encoding polypeptides according to theinvention preferably include a secretion sequence in order to facilitatesecretion of the polypeptide from bacterial hosts, such that it will beproduced as a soluble native peptide rather than in an inclusion body.The peptide may be recovered from the bacterial periplasmic space, orthe culture medium, as appropriate.

Suitable promoting sequences for use with yeast hosts may be regulatedor constitutive and are preferably derived from a highly expressed yeastgene, especially a Saccharomyces cerevisiae gene. Thus, the promoter ofthe TRP1 gene, the ADHI or ADHII gene, the acid phosphatase (PH05) gene,a promoter of the yeast mating pheromone genes coding for the a- ora-factor or a promoter derived from a gene encoding a glycolytic enzymesuch as the promoter of the enolase, glyceraldehyde-3-phosphatedehydrogenase (GAP), 3-phospho glycerate kinase (PGK), hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphateisomerase, phosphoglucose isomerase or glucokinase genes, the S.cerevisiae GAL 4 gene, the S. pombe nmt 1 gene or a promoter from theTATA binding protein (TBP) gene can be used. Furthermore, it is possibleto use hybrid promoters comprising upstream activation sequences (UAS)of one yeast gene and downstream promoter elements including afunctional TATA box of another yeast gene, for example a hybrid promoterincluding the UAS(s) of the yeast PH05 gene and downstream promoterelements including a functional TATA box of the yeast GAP gene (PH05-GAPhybrid promoter). A suitable constitutive PHO5 promoter is e.g. ashortened acid phosphatase PH05 promoter devoid of the upstreamregulatory elements (UAS) such as the PH05 (−173) promoter elementstarting at nucleotide-173 and ending at nucleotide-9 of the PH05 gene.

Gene transcription from vectors in mammalian hosts may be controlled bypromoters derived from the genomes of viruses such as polyoma virus,adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus,cytomegalovirus (CMV), a retrovirus, and Simian Virus 40 (SV40), fromheterologous mammalian promoters such as the actin promoter or a verystrong promoter, e.g. a ribosomal protein promoter, and from thepromoter normally associated with the naturally occurring sequenceencoding the polypeptide at issue, provided such promoters arecompatible with the host cell systems.

Transcription of a DNA encoding a polypeptide according to the inventionby higher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are relatively orientation and positionindependent. Many enhancer sequences are known from mammalian genes(e.g. elastase and globin). However, typically one will employ anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270) and theCMV early promoter enhancer. The enhancer may be spliced into the vectorat a position 5′ or 3′ to the coding sequence, but is preferably locatedat a site 5′ from the promoter.

Advantageously, a eukaryotic expression vector encoding a polyeptideaccording to the invention may comprise a locus control region (LCR).LCRs are capable of directing high-level integration site independentexpression of transgenes integrated into host cell chromatin, which isof importance especially where the gene is to be expressed in thecontext of a permanently-transfected eukaryotic cell lint in whichchromosomal integration of the vector has occurred, in vectors designedfor gene therapy applications or in transgenic animals.

Eukaryotic expression vectors will also contain sequences necessary forthe termination of transcription and for stabilising the mRNA. Suchsequences are commonly available from the 5′ and 3′ untranslated regionsof eukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the polypeptide according to the invention.

An expression vector includes any vector capable of expressing nucleicacids that are operatively linked with regulatory sequences, such aspromoter regions, that are capable of expression of such DNAs. Thus, anexpression vector refers to a recombinant DNA or RNA construct, such asa plasmid, a phage, recombinant virus or other vector, that uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those withordinary skill in the art and include those that are replicable ineukaryotic and/or prokaryotic cells and those that remain episomal orthose which integrate into the host cell genome. For example, DNAsencoding a polypeptide according to the invention may be inserted into avector suitable for expression of cDNAs in mammalian cells, e.g. a CMVenhancer-based vector such as pEVRF (Matthias, et al., (1989) NAR 17,6418).

Particularly useful for practising the present invention are expressionvectors that provide for the transient expression of DNA encodingpolypeptides according to the invention in mammalian cells. Transientexpression usually involves the use of an expression vector that is ableto replicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector, and, in turn,synthesises high levels of polypeptides according to the invention. Forthe purposes of the present invention, transient expression systems areuseful e.g. for identifying mutants of polypeptides according to theinvention, to identify potential phosphorylation sites, or tocharacterise functional domains of the protein.

Construction of vectors according to the invention employs conventionalligation techniques. Isolated plasmids or DNA fragments are cleaved,tailored, and religated in the form desired to generate the plasmidsrequired. If desired, analysis to confirm correct sequences in theconstructed plasmids is performed in a known fashion. Gene presence,amplification and/or expression may be measured in a sample directly,for example, by conventional Southern blotting, Northern blotting toquantitate the transcription of mRNA, dot blotting (DNA or RNAanalysis), or in situ hybridisation, using an appropriately labelledprobe which may be based on a sequence provided herein. Those skilled inthe art will readily envisage how these methods may be modified, ifdesired.

In accordance with another embodiment of the present invention, thereare provided cells containing the above-described nucleic acids. Suchhost cells such as prokaryote, yeast and higher eukaryote cells may beused for replicating DNA and producing polypeptides according to theinvention. Suitable prokaryotes include eubacteria, such asGram-negative or Gram-positive organisms, such as E. coli, e.g. E. coliK-12 strains, DH5α (and HB101, or Bacilli. Further hosts suitable forpolypeptides according to the invention encoding vectors includeeukaryotic microbes such as filamentous fungi or yeast, e.g.Saccharomyces cerevisiae. Higher eukaryotic cells include insect andvertebrate cells, particularly mammalian cells, including human cells,or nucleated cells from other multicellular organisms. In recent yearspropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure. Examples of useful mammalian host cell lines areepithelial or fibroblastic cell lines such as Chinese hamster ovary(CHO) cells, NIH 3T3 cells, HeLa cells or 293T cells. The host cellsreferred to in this disclosure comprise cells in in vitro culture aswell as cells that are within a host animal.

DNA may be stably incorporated into cells or may be transientlyexpressed using methods known in the art. Stably transfected mammaliancells may be prepared by transfecting cells with an expression vectorhaving a selectable marker gene, and growing the transfected cells underconditions selective for cells expressing the marker gene. To preparetransient transfectants, mammalian cells are transfected with a reportergene to monitor transfection efficiency.

To produce such stably or transiently transfected cells, the cellsshould be transfected with a sufficient amount of nucleic acid encodingpolypeptides according to the invention. The precise amounts of suchnucleic acid may be empirically determined and optimised for aparticular cell and assay.

Host cells are transfected or, preferably, transformed with theabove-captioned expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. Heterologous DNA may be introduced intohost cells by any method known in the art, such as transfection with avector encoding a heterologous DNA by the calcium phosphatecoprecipitation technique or by electroporation. Numerous methods oftransfection are known to the skilled worker in the field. Successfultransfection is generally recognised when any indication of theoperation of this vector occurs in the host cell. Transformation isachieved using standard techniques appropriate to the particular hostcells used.

Incorporation of cloned DNA into a suitable expression vector,transfection of eukaryotic cells with a plasmid vector or a combinationof plasmid vectors, each encoding one or more distinct genes or withlinear DNA, and selection of transfected cells are well known in the art(see, e.g. Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press).

Transfected or transformed cells are cultured using media and culturingmethods known in the art, preferably under conditions, wherebypolypeptides according to the invention encoded by the DNA is expressed.The composition of suitable media is known to those in the art, so thatthey can be readily prepared. Suitable culturing media are alsocommercially available.

Labelling of Polypeptides

Many amino acid residues have chemistry allowing labelling withcommercially available fluorescent and other labels. The mostsignificant of these are those with ionizable side chains—aspartic acid,glutamic acid, lysine, arginine, cysteine, histidine and tyrosine. Thelabelling reagent will comprise a group conferring the desired propertysuch as fluorescence and a group involved in the conjugation of label totarget. The most commonly used functional groups in this context arethose which react with amines by either an acylation or alkylationroute. These include isothiocyanates, isocyanates, acyl azides, NHSesters and many others. Also common is the use of thiol-directed groupssuch as haloacetates and maleimides. These label primarily at the freesulfhydryl group of a cysteine residue.

A number of protocols have been devised to achieve labelling at aspecific site in a synthesised peptide. These are available in theliterature and many are detailed in Bioconjugate Techniques, G. T.Hermanson, Academic Press 1996. It is important to note that, while itis possible to bias reactions to achieve specific labelling on onefunctional group and to prevent promiscuous reaction of the label withother sites in the peptide or protein, it is unlikely to be possible tolabel, for example, one lysine specifically in a peptide containingmultiple amine groups. To achieve this, labelling of the molecule ispreferably concurrent with synthesis. This may be achieved either by theuse of a labelled amino acid in the synthesis process or by the specificdeprotection and labelling of the residue of interest beforedeprotection of other potentially reactive residues at the completion ofthe synthesis.

Incubation of Polypeptides in Assays According to the Invention

Buffer/ionic strength—the incubation solution should comprise anappropriate buffer (as defined above) and should be of an ionic strengthsuitable for the formation of a folded reporter molecule structure(0-150 mM of a suitable salt).

Concentration—peptides should be present at a concentration sufficientlyhigh to allow formation of the folded reporter molecule and detection ofthe assay output (within the limits of instrumentation) but not so highthat the detector is saturated.

Temperature—the temperature selected will be suitable for both formationof the reporter multimer and also activity of any biological agentsincluded in the assay (i.e. 4-40° C.).

Note that each of these parameters are preferably empirically determinedas the behaviour of the reporter molecule will be sequence dependent.

The invention is described below, for the purpose of illustration only,in the following examples. Modifications of the techniques describedherein will be apparent to those skilled in the art.

General Methods Useful for the Detection of Proteolysis

-   Purification of proteolytic enzymes-   Synthesis of coiled coil peptides-   Labelling coiled coil peptides with fluorophores-   Purification of fluorescent peptides-   Proteolysis of peptides in vitro-   Fluorescence measurement of proteolysis in vitro in real time-   Reporter group proteolysis in living cells-   Heterologous expression of peptides.    Purification of Proteolytic Enzymes

The proteases described can be purified from natural sources or fromcells/organisms engineered to heterologously express the enzymes. Allenzymes used to illustrate the current invention are availablecommercially. Details of the purifications from natural sources areshown in table 3. Purification from a recombinant source can be achievedby one of several standard methods including the use of a histidine tagas an extension to the protein for purification on a nickel chelatingaffinity column (used for purification of TEV protease).

TABLE 3 Protease Source Reference Chymotrypsin bovine pancreas Thrombinbovine plasma TEV recombinant, E. coli Life Technologies Ltd. (histidinetag) Aminopeptidase M porcine kidney Carboxypeptidase P Penicilliumjanthinellum Carboxypeptidase Y yeast Hayashi, R., Moore, S. & Stein, W.H., Journal of Biological Chemistry (1973) 248 2296-2302

Synthesis of Coiled Coil Peptides

Coiled coil peptides are synthesised by Fmoc or Tboc chemistry accordingto the methods of Atherton, E., Logan, C. J., & Sheppard (1981), J.Chem. Soc. 1981 538-546 and Perkin, I. & Merrifield R. B. (1963) J. Am.Chem. Soc. 85, 2149-2154 respectively. Following deprotection andcleavage from the resin, peptides are desalted by gel filtrationchromatography and analysed by mass spec, HPLC and Edman degradationsequencing using standard methodologies.

Labelling Coiled Coil Peptides With Fluorophores

Coiled coil peptides are labelled with thiol reactive or primary aminereactive derivatives of fluorescein and tetramethylrhodamine or othersuitable FRET partners (see table 4; Molecular Probes, Eugene, Oreg.,USA) using procedures described by Hermanson, G. T. (1995) BioconjugateTechniques, Academic Press, London.

TABLE 4 Donor Acceptor R₀ (Å) Fluorescein Tetramethylrhodamine 55 IEDANSFluorescein 46 (5-((((2-iodoacetyl)- amino)ethyl)amino)- naphthalene-1-sulfonic acid) EDANS (5-((2-aminoethyl)- DABCYL (4-((4- 33amino)naphthalene- (dimethylamino)phenyl) 1-sulfonic acid) azo)benzoicacid) Fluorescein Fluorescein 44 BODIPY ™ FL (4,4- BODIPY ™ FL 57difluoro-5,7-dimethyl-4- bora-3a,4a-diaza-s- indacene-3-propionic acid)

Purification of Fluorescent Peptides

Fluorescent peptides are separated from unreacted fluorophores by gelfiltration chromatography or reverse phase HPLC.

Proteolytic Cleavage of Peptides

Chymotrypsin—Lyophilised chymotrypsin is dissolved in 1 mM HCl. Peptides(0.001-100 μM) are cleaved by chymotrypsin (1:200-1:20 w/w) in either100 mM Tris-HCl pH 7.5, 12 mM NaCl, 10 mM CaCl₂ or 50 mM HEPES pH 7.0,120M NaCl, 10 mM CaCl₂, at 1-40° C. for periods of time ranging from 0to 24 hours.

Thrombin—Peptides (0.001-100 μM) are cleaved by thrombin (1:100-1:10w/w) in either 50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 2.5 mM CaCl₂, (0.1%2-mercaptoethanol if required); or 50 mM HEPES pH 7.0, 120 mM NaCl, 2.5mM CaCl₂ at 1-40° C. for periods of time ranging from 0 to 24 hours.

Aminopeptidase M—Peptides (0.001-100 μM) are cleaved by Aminopeptidase M(1:50-1:500 w/w) in 42.2 mM K₂HPO₄, 7.8 mM KH₂PO₄, pH 7.0, 120 mM NaCl,at 1-40° C. for periods of time ranging from 0 to 24 hours.

Carboxypeptidase Y—Peptides (0.001-100 μM) are cleaved byCarboxypeptidase Y (1:200-1:20 w/w) in 50 mM sodium citrate pH 6.0, 120mM NaCl, or 42.2 mM K₂HPO₄, 7.8 mM KH₂PO₄, pH 7.0, 120 mM NaCl at 1-40°C. for periods of time ranging from 0 to 24 hours.

Carboxypeptidase P—Peptides (0.001-100 μM) are cleaved byCarboxypeptidase P (1:100-1:10) in 50 mM sodium citrate pH 4.0, 120 mMNaCl; or 42.2 mM K₂HPO₄, 7.8 mM KH₂PO₄, pH 7.0, 120 mM NaCl at 1-40° C.for periods of time ranging from 0 to 24 hours.

TEV-Peptides (0.001-100 μM) are cleaved by TEV (1:300-1:30 w/w) in 50 mMTris-HCl (pH 7), 0.5 mM EDTA, 1 mM DTT, 120 mM NaCl; or 42.2 mM K₂HPO₄,pH 7.0, 7.8 mM KH₂PO₄, 0.5 mM EDTA pH 7.0 at 1-40° C. for periods oftime ranging from 0 to 24 hours.

In each case reactions are monitored in a 1 cm cell in a JASCO 715spectropolarimeter, the CD at 222 m was observed to follow any change instructure of the peptides present (Circular Dichroism and Optical rotaryDispersion of proteins and polypeptides, Alder, A. J., Greenfield, N. J.& Fasman G. D., Methods in Enzymology (1973) 27 675-796). To confirm thetime course of the reaction samples were quenched in 1 mM PMSF or byother suitable means and analysed by mass spectrometry using standardmethods.

Fluorescence Measurements of Proteolysis in Vitro in Real Time

Fluorophore labelled coiled coil peptide's (in a 1:6 molar ratio offluorescein-labelled (pepF) to tetramethylrhodamine-labelled (pepR)peptide) are mixed. Samples are analysed in a fluorimeter usingexcitation wavelengtbs relevant to pepF (˜450 nm) and emissionwavelengths relevant to pepF (˜516 nm) and pepR (˜580 nm). A ratio ofemission from pepR over that from pepF following excitation at a singlewavelength is used to determine the efficiency of FRET betweenfluorophores, and hence their spatial proximity. Typically themeasurements are performed at 0-37° C. as a function of time followingthe addition of the relevant protease in an appropriate buffer asdetailed above.

Reporter Group Proteolysis in Living Cells

PepF:PepR (where pepF and pepR represent reporter sequences specific forcaspase activity) are microinjected into live cells (e.g. Jurkat or HeLacells). The ratio of emission from pepR/pepF is measured as describedabove via a photomultiplier tube focused on a single cell. Induction ofapoptosis is achieved by treating with anti-Fas monoclonal antibody orTNF-α plus cycloheximide respectively as described in Cryns et al., J.Biol. Chem (1996) 271 31277-31282. Caspase activity is monitored as achange in the ratio of pepR/pepF fluorescence.

Heterologous Expression of Peptides

Coiled coil peptides can be synthesised from the heterologous expressionof cDNA sequences for coiled coil domains of interest modified toinclude the sequence for proteolytic modification as appropriate, orsynthetic gene of the same. Expression can be in prokaryotic oreukaryotic cells using a variety of plasmid vectors capable ofinstructing heterologous expression. Purification of these products isachieved by destruction of the cells (e.g. French Press) andchromatographic purification of the products. This latter procedure canbe simplified by the inclusion of an affinity purification tag at oneextreme of the peptide, separated from the peptide by a proteasecleavage site (other than that of interest) if necessary.

Heterologous expression of the peptides as described above can also beadapted to facilitate in vitro or in vivo assay of proteolytic events.The cDNA or synthetic gene used would code for the peptide to beexpressed fused to a variant of GFP, at the N or C terminus. Twoplasmids or a plasmid carrying two genes would be introduced such thattwo peptides are expressed, capable of forming a dimer and fused withdifferent GFP variants capable of FRET. Thus, on expression of the twopeptides, a dimer would form and FRET would be observed. Cleavage eventswould destroy the dimer and FRET would be lost. The expression of thepeptides in the cell to be investigated removes the need formicroinjection of the reporter peptides.

EXAMPLE 1

Chymotrypsin Assay

Chymotrypsin is a digestive enzyme found in the small intestine. Thecoiled coil peptide of GCN4 can be adapted to provide a homodimericreporter molecule able to monitor cleavage by chymotrypsin in the mannerdescribed above.

The position of the labelling site is determined as follows—

The label must be in a position where:

-   -   it does not interfere substantially with the assembly of the        reporter molecule at the reaction temperature (in the coiled        coil, modifications to positions b, c or f are least disruptive;        positions e and g are more disruptive; and positions a and d are        most disruptive to coil formation)    -   it is sufficiently far from the site of chemical modification        not to interfere with enzyme activity    -   it is in a highly structured region of the reporter to ensure        that the position and orientation of the labels with each other        can be well understood (in a coiled coil this preferably entails        avoiding a position in the terminal heptads)    -   the labels are as close in space as is possible given other        constraints.

An assessment of potential locations is made using any structuralinformation available and also homology with other known structures.

Peptide A is synthesised, incorporating a C residue at position 273 forlabelling purposes:

RMKQLEDKVEELLSKTY-HLENEVACLKKLVGERAAK Peptide A (SEQ ID NO:24)

This sequence is derived from that of amino acids 249-281 of GCN4(Genbank Accession No. K02205.

(X-X represents the protease cut site, Y residue in bold indicates therecognition feature of the enzyme).

N₂₆₄ has been changed to T, and R₂₇₃ has been changed to C. The tyrosineresidue (shown here in bold type) is the residue at which chymotrypsincleaves. The cysteine residue (also shown in bold type) provides thesite for attachment of thiol-directed fluorescent labels.

The circular dichroism (measured in units of ellipticity) of proteins at222 nm provides a measure of the amount of α-helix present in thestructure, with a large, negative ellipticity indicting a high level ofhelicity. The coiled-coil has a distinctive α-helical CD spectrum withminima at 222 nm and 208 nm (O'Shea et al., 1989, Science, 243:538-542). A timecourse of peptide A cleavage by chymotrypsin (20:1 w/w)is followed by digesting 10 μM peptide A in 50 mM Tris-HCl pH 7.5, 50 mMNaCl, 10 mM CaCl₂ at 30° C. in a JASCO 715 spectropolarimeter andobserving the change in θ at 222 nm. A wavelength scan (260-200 nm) isalso taken of the sample at the start and end of the experiment. Thesedata show that peptide cleavage by chymotrypsin results in the loss ofcoiled coil structure as required by the assay, supporting thefluorimetric data presented below.

A time-course digestion of Peptide A with chymotrypsin is analysed byMALDI-TOF mass spectrometry. On the protein matrix (MS, denoted SA oneach spectrogram) the Peptide A substrate as 4245Da is evident. It isdegraded completely over two hours to a fragment of 2155 Da (which isthe N-terminal fragment, cleaved at the Y—H bond). The C-terminalfragment appears to be cleaved at a second site (after L-26) generatinga product of 1100 Da, although the final peptide fragment of about 990Da is not observed in the MS.

Peptides are then labelled using a method adapted from one known in theart (Hermanson, 1997, Bioconjugate Techniques, Academic Press). 20 mMfluorescein iodoacetamide (IAF), or 20 mM tetramethylrhodamineiodoacetamide (TMRIA) in DMSO and 0.23 mM peptide in 20 mM TES buffer,pH 7.0 are prepared. Fluorophore and peptide are mixed in a molar ratioof 0.9:1 (label:peptide) and incubated at 4° C. in the dark for aminimum of 2 hours. Labelling is assessed by reverse phase HPLC (C18column; solvent A: H₂O/0.1% TFA; solvent B: acetonitrile/0.1% TFA) andMALDI-TOF mass spectrometry. Peptide A labelled with fluorescein(Peptide AF) and rhodamine (Peptide AR) are thus generated.

FRET occurs between fluorophores attached to undigested peptides, whichinteract with each other, but not between digested peptides, which donot interact (evidenced by loss of structure in CD experiments). Onlythe fluorescence emission quench of the donor fluorophore is displayedin these experiments.

Fluorescence at 516 nm is measured for labelled peptide A in a 1 cmpathlength cell at peptide concentrations of 0.08 μM peptide AF and 0.48μM peptide AR in 50 mM HEPES pH 7.0, 150 mM NaCl, 10 nM CaCl₂ at 30° C.in a PTI fluorimeter system with temperature controlled by a waterbath.Upon addition of peptide AR to peptide AF, fluorescence in the region offluorescein emission decreases. This suggests that energy transfer istaking place.

A timecourse of peptide A cleavage by chymotrypsin is followed bydigesting 8 μM AF and 48 μM AR in 50 mM HEPES pH 7.0, 150 mM NaCl, 10 mMCaCl₂ at ˜30° C. Samples are removed at 10 minute intervals and diluted100×into 50 mM HEPES pH 7.0, 150 mM NaCl, 10 mM CaCl₂, and an emissionscan (excitation wavelength 450 nm) is recorded at ˜30° C. for eachdiluted sample. This data is presented as a ratio of emission 580nm/emission 515 nm to eliminate any concentration differences introducedon dilution of the samples. A decrease in this ratio represents loss ofFRET which is correlated with peptide cleavage. The fluorometric datagathered over this timecourse corresponds well with that observed byMALDI-TOF, a plateau being reached by 2 hours which is the time point atwhich the mass spectrometric data.

In addition, no FRET is observed when the cleaved fluorescein labelledpeptide A (peptide ADF) is mixed with peptide AR, indicating that wheneven one polypeptide partner is digested, formation of the coiled coilstructure and, hence, protein:protein heterodimerisation (with respectto fluorophore composition) cannot occur.

Together, these results, that FRET does not occur between the digestedmolecules or between a digested and an undigested molecule, indicatedthat FRET using labelled, polypeptides comprising a coiled-coil, may beused successfully in the invention to report on the digestion ofpolypeptides by proteolytic enzymes.

EXAMPLE 2

Amino- and Carboxypeptidases

Exopeptidases are important in the breakdown of peptides after initialdigestion by endopeptidases such as chyrnotrypsin or trypsin. The endproduct of attack by such enzymes are free amino acids or dipeptides. Anassay for this type of proteolysis relies upon the fluorophores beingfar from the initial site of proteolysis such that digestion ultimatelyresults in a fragment which is too small to remain within the coiledcoil multimer, at which point FRET is lost. An unblocked N-terminus orC-terminus is essential for the activity of exopeptidases.

Artificial designer peptides are synthesised such that a heterodimericcoiled coil is formed upon mixing of the partners.

IAALRERICYLRERNQQLRQRIQQL peptide B (SEQ ID NO:25)acetyl-IAALEREIYKLEQENQQLEQEIQQL-amide peptide C (SEQ ID NO:26)

Peptides B and C are labelled as set forth in example 1, to produce BFand CR (or vice versa), labelled with fluorescein and rhodaminerespectively.

Upon mixing, FRET is observed as heterodimers are formed. When peptidesBF and CR are incubated in the presence of aminopeptidase M orcarboxypeptidase Y, together with an appropriate buffer, FRET is lostdue to digestion of the coiled coil to a point where structure can nolonger be maintained.

EXAMPLE 3

TEV (Tobacco Etch Virus) Protease

This protease is important in the processing of the precursorpolyprotein of TEV (Mutational analysis of Tobacco Etch Viruspolyprotein processing: cis and trans proteolytic activities ofpolyproteins containing the 49-kilodalton proteinase, Carrington, J. C.,Cary, S. M. & Dougherty, W. G., Journal of Virology (1988) 622313-2320). In the laboratory it is commonly used in the cleavage ofaffinity tags from recombinant proteins after the purification process(Release of proteins and peptides from fusion proteins using arecombinant plant virus proteinase, Parks, T. D., Leuther, K. K.,Howard, E. D., Johnston, S. A. & Dougherty, W. G., AnalyticalBiochemistry (1994) 216 413-417). The seven amino acid recognition siteallows highly specific cleavage and makes undesirable cleavage of therecombinant protein unlikely.

RMKQLEDKVENLYSQ-SYHLENEVACLKKLVGER Peptide 2 (SEQ ID NO:27)

Peptide 2 is constructed to contain the TEV protease cleavage site, andlabelled at an inserted unique cysteine residue, as for Peptide A. Thelabelling and FRET assay of Example 1 is repeated, with identicalresults.

EXAMPLE 4

Thrombin

Thrombin is the final enzyme in the blood clotting cascade, cleavingfibrinogen to fibrin. Its effects are, in part, regulated by a negativefeedback mechanism in which thrombin activates protein C whichultimately switches off the cascade and therefore thrombin production.This enzyme is also important in promoting mitosis in fibroblasts,chemotaxis in monocytes and neurite retraction in neurons. The coiledcoil peptide of GCN4 can be adapted to provide a homodimeric reportermolecule to monitor cleavage by thrombin in the manner described above.In order to have cleavage at one site only it is necessary to eliminateall arginine residues other than that at the intended cleavage site fromthe peptide (as in Peptide 3 below).

KMKQLEDKVR-ELLSKNYHLENEVACLLKKLVGER Peptide 3 (SEQ ID NO:28)

The procedure of Example 1 is repeated with Peptide 3, and againidentical results are obtained.

EXAMPLE 5

Caspases

The caspases (Caspases: enemies within, Thornberry, N. A. & Lazebnik, Y.(1998) Science 281 1312-1316) are a family of proteases with an absoluterequirement for cleavage after aspartic acid. They are synthesised asinactive proenzymes and cleaved either autocatalytically or by anothermember of the family to form an active heterodimer of a large and asmall subunit. This family of enzymes play a role in inflammation but,more importantly, several members of the family provide signalstriggering apoptosis of the cell and others are directly involved incell disassembly itself. The role of caspases in the apoptotic processincludes the inactivation of proteins protective against apoptosis suchas the Bcl-2 proteins, the destruction of proteins with a key role incell structure such as the lamins and the deregulation of proteins bythe disruption of links between regulatory and effector domains. A lossof control of apoptosis leading either to excessive or inadequate celldeath plays a role in many disease processes including cancers,neurodegenerative disorders and auto-immune disease. Evidence is alreadyavailable suggesting that caspase inhibitors are able to protect againstinappropriate cell death.

The coiled coil peptide of GCN4 is adapted to provide a homodimericreporter molecule to monitor cleavage by members of the caspase family(e.g. for Caspase 2, 3, 7 below) in the manner described above.

RMKQLEDKVEELLDEND-HLENEVACLKKLVGER Peptide 4 (SEQ ID NO:29)

The procedure of Example 1 is repeated with Peptide 4, and identicalresults are obtained.

EXAMPLE 6

Fatty Acylation and GPI Anchor Attachment

As discussed above, the assay format according to the invention can beextended to measure post-translational modification events which haveproteolysis as an integral step. The molecular basis of these assays isthe destabilisation of the binding partnership following the proteolyticevent which, if coincident with the covalent addition of another group(GPI, farnesyl etc.), provides an indirect reporter function for theseclasses of post-translational modification.

Fatty acylation of proteins is a dynamic post-translational modificationwhich is critical for the biological activity of many proteins, as wellas their interactions with other proteins and with membranes. Thus, fora large number of proteins, the location of the protein within a cellcan be controlled by its state of prenylation (fatty acid modification)as can its ability to interact with effector enzymes (ras and MAPkinase, Itoh, T., Kaibuchi, T., Masuda, T., Yamamoto, T., Matsuura, Y.,Maeda, A., Shimizu, K., & Takai, Y. (1993) J. Biol. Chem. 268, 3025-;ras and adenylate cyclase (in yeast) Horiuchi, H., Kaibuchi, K.,Kawamura, M., Matsuura, Y., Suzuki, N., Kuroda, Y., Kataoka, T., &Takai, Y. (1992) Mol. Cell. Biol. 12, 4515-) or with regulatory proteins(Shirataki et al., 1991, above). The prenylation status of ras isimportant for its oncogenic properties (Cox, 1995, above) thusinterference with the prenylation status of ras is considered a valuableanti-cancer strategy (Hancock, J. F. (1993) Current Biology 3, 770)

The coiled coil of GCN4 is adapted to produce a homodimeric reportermolecule capable of monitoring geranylgeranylation according to theprocedure described in Example 1. The adaptations are (i) to shorten thelength of the coiled-coil structure to achieve a coiled coil oligomer ofreasonable stability; (ii) to modify the C-terminal residues asnecessary to comply with the substrate recognition features ofgeranylgeranyltransferase I (GGT I).

KVEELLSKNYHLENEVARLK C ALL Peptide 5 (SEQ ID NO:30)

The recognition site is detailed in bold, the underlined cysteine is thesite of geranylgeranylation. The concomitant proteolytic removal of theC-terminal residues (ALL) will result in destabilisation of thecoiled-coil structure and will prompt dissociation of the oligomer. Thiscan be used as an indirect measure of fatty acylation. A change in theC-terminal amino acid (to Ser, Met, Ala or Gln) alters the specificityof this reporter to measure farnesylation (Protein prenyltransferases,Casey, P. J & Seabra, M. C., Journal of Biological Chemistry (1996) 2715289-5292).

Labelling for FRET is achieved in two ways: by N-terminal capping andmutation of the existing K to R, thus providing a primary amine to whicha fluorophore may be attached, or by incorporation of fluorescentresidues during peptide synthesis (see above).

Peptide 5 is labelled with fluorescein or rhodamine, and assayed forFRET under the conditions of Example 1. Geranylgeranylation is thenperformed by incubating 1-4 μg of the reporter molecule with 25 μlrabbit reticulocyte lysate (or the equivalent quantity of either apurified geranylgeranyltranferase or a test sample) and geranylgeranylpyrophosphate (40 μM) in a final volume of at least 50 μl (Cox 1995,above). The modification of the reporter is followed by observation ofacceptor/donor fluorescence emission upon excitation of the donorfluorophore at an appropriate wavelength. A decrease in this ratioindicates a loss of FRET which is a measure of the geranylgeranylationof the reporter.

A number of membrane proteins are anchored in the membrane through thecovalent attachment of a glycosylated phospholipid, most commonlyglycosylphosphatidyl-inositol (GPI), to the C-terminus of thepolypeptide (reviewed in Udenfriend, S., & Kodukula, K. (1995) Howglycosyl-phosphatidylinositol-anchored membrane proteins are made. Ann.Rev. Biochem. 64, 563-591). These proteins do not have a transmembranedomain made of amino acids, instead covalent attachment of aglycosylated form of phosphatidyl inositol is attached to the C-terminalresidue of the protein (reviewed in Udenfriend & Kodukula, 1995;Kinoshita, T., Ohishi, K., & Takeda, J., (1997) GPI-anchor synthesis inmammalian cells: Genes, their products and a deficiency. J. Biochem.(Tokyo) 122, 251-257). This anchors the protein in the (usually plasma)membrane. This form of membrane association permits the release of thepolypeptide from the membrane following the hydrolysis of the anchor bythe enzyme phospholipase C (or phospholipase D). In the case of plasmamembrane associated GPI-anchored proteins this will release the proteinfrom the cell into the blood stream or extracellular solution.Approximately 50 proteins are known to be attached to membranes by thisroute, including prion proteins, acetylcholinesterase, alkalinephosphatase, folate transporter, urokinase receptor, cell adhesionmolecules (N-CAM), trypanosome antigenic proteins (variant surfaceglycoproteins, malaria), CD-24 (small cell lung carcinoma antigen)(reviewed in Udenfriend & Kodukula, 1995). This can facilitate theshedding of protein antigen (by parasites) to avoid immunodetection.

The attachment of the GPI anchor occurs in the lumen of the ER, andoccurs within one minute of protein synthesis in living cells. Proteinstargeted for GPI anchoring have at least two primary sequence signals,(i) an N-terminal leader sequence to facilitate their transport into theER, and (ii) a C-terminal sequence which directs GPI anchor attachment.There is some considerable similarity between these two signals (Yan,W., Shen, F., Dillon, B., & Ratnam, M. (1998) The hydrophobic domains inthe carboxyl-terminal signal for GPI modification and in theamino-terminal leader peptide have similar structural requirements. J.Mol. Biol. 275, 25-33.) A schematic representation of the proteinstructure is as follows:

The aim of this assay is to produce a substrate which oligomerises usinga coiled coil interaction, prior to, but not following GPI anchorattachment. The structural feature responsible for oligomerisation musttherefore reside within the C-terminal signal sequence for GPIattachment.

A construct is used consisting of the leader sequence and part of thefolate receptor (Genbank Accession No: X69516) followed by an artificialsequence containing the consensus sequence for GPI anchor addition whichadopts a heteromultimeric coiled-coil (FIG. 6).

In FIG. 6, amino acids in bold represent the GPI anchor attachment site;the underlined sequence is the hydrophilic region and the sequence initalics is the hydrophobic region of the recognition site; X representsthe amino acid used for measuring the reporter output, K maybederivatised with dansyl.

Fluorescently labelled reporter molecules are produced and incorporatedin an assay for GPI anchor addition using methods derived from Crowley,K. S., Reinhart, G. D. and Johnson, A. E. (1993) Cell 73, 1101-1115 andKodukula, K., Micanovic, R., Gerber, L., Tamburrini, M., Brink, L. andUdenfriend, S. (1991) Journal of Biological Chemistry 266, 4464-4470.

Isolation of tRNA^(lys) from 10 mg of unfractionated brewer's yeast tRNAis achieved using fast protein liquid chromatography (FPLC) with a MonoQHR 10/10 column. Elution was in 10 mM MgCl, 10 mM sodium acetate (pH4.5) with a linear gradient of NaCl from 0.48M to 11.0M. Fractionsenriched in tRNA^(lys) are identified by aminoacylation assays and thenucleic acid precipitated in ethanol. After dialysis the fractions arerepurified by FPLC as before and fractions with the highest level oftRNA^(lys) are precipitated, dialysed and aminoacylated with lysine. Theaminoacylated tRNA^(lys) is purified by FPLC, precipitated and dialysedinto 1 mM potassium acetate (pH5.0) at 4° C. This is stored at −75° C.

tRNA^(lys) prepared as above has been shown to be selectively modifiedat the ε amino group with NBD using the method of Johnson et al(Johnson, A. E., Woodward, W. R., Herbert, E. and Menninger, J. R.(1976) N ^(e)-acetllysine transfer ribonucleic acid; a biologicallyactive analogue of aminoacyl transfer ribonucleic acids. Biochemistry15, 569-575). We therefore use a parallel method to label tRNA^(lys)with a dansyl group (a good FRET partner for tryptophan).6-((5-dimethylaminonaphthalene-1-sulfonyl)amino)hexanoic acid,succinimidyl ester (dansyl-X-SE; Molecular Probes) (9 mg) is solubilizedin 1.75 ml of dimethylsulphoxide and added to 5 nmol lys-tRNA in 750 mlof 50 mM potassium phosphate (pH7.0). The labelling reaction isinitiated by the addition of 15 μl of freshly made 4M KOH while stirringvigorously. The reaction is allowed to proceed for 20 seconds at 20° C.before the addition of 151 of 4M acetic acid to terminate the reaction.The tRNA is immediately ethanol precipitated. Excess free dye is removedby redissolving the pellet and ethanol precipitating the product for asecond time. This method yields a high proportion of labelled lys-tRNAwhich is purified by FPLC using a benzoylated diethylaminoethylcellulose column. The column is washed with 1M NaCl, 10 mM MgCl₂, 10 mMsodium acetate (pH 4.5) and then eluted with 25 ml of 2M NaCl, 10 mMMgCl₂, 10 mM sodium acetate (pH4.5), 25% (v/v) ethanol. Thedansyl-lys-tRNA is ethanol precipitated, resuspended and dialysed into 1mM potassium acetate for storage.

Plasmids are prepared carrying the coding sequence for the reporterdescribed above using well known and published methods. The plasmids arelinearised and transcribed using a Riboprobe-II kit (Promega Biotech) togenerate mRNA for in vitro translation.

Rough microsomal membranes (RM) are prepared from Chinese hamster ovary(CHO) cells. Cells are maintained in Iscove's modified Dulbecco's mediumwith 10% foetal calf serum (FCS) and are harvested at confluency, washedtwice with ice-cold phosphate buffered saline (PBS) and scraped intoPBS. These cells are pelleted at 1000×g and washed twice with PBS.Pelleted cells are resuspended in 10 mM Tris (pH7.5) and incubated onice for 5 minutes. Maintaining a ratio of 1:15 (cells:buffer), the cellsuspension is diluted with an equal volume of 600 mM sucrose, 6 mMdithiothreitol and incubated for a further 5 minutes on ice. Thissuspension is homogenised with 10 strokes in a Dounce homogeniser at 4°C. The homogenate is centrifuged for 2×10 minutes at 7700×g and ×20minutes at 17,300×g. In each case the pellet is discarded.Centrifugation of the final supernatant at 100,000×g for 60-75 minutesyields rough microsomal membranes.

The fluorescently labelled dansyl-lys-tRNA is used together with an mRNAtranscription mix for in vitro translation of the reporter peptides inthe presence of rough microsomal membranes using a translation systemsuch as the rabbit reticulocyte lysate supplied by Promega. A 25 μltranslation mixture is prepared containing 12.5 μl nuclease-treatedrabbit reticulocyte lysate, 0.04 mM mixed amino acids (minusmethionine), 12-15 pmol dansyl-lys-tRNA, 20 units RNase inhibitor,1.5-2.0 μl [³⁵S]methionine (15 mCi/ml; 1100 Ci/mmol) and 1-2 μg reportermRNA. This mixture is preincubated for 2 min at 30° C. before theaddition of RM.

RM are resuspended by repetitive pipetting in translation buffer (100 mMKCl, 4 mM Mg²⁺, 50 mM sucrose, 3 mM dithiothreitol and a cocktail ofprotease inhibitors including aprotinin, antipain, bestatin,chymostatin, leupeptin and pepstatin each at 2 μg/ml and RNase inhibitorat 5 units/ml) and 5 μl of this membrane suspension is added to thetranslation mixture.

Translations are incubated at 20-30° C. for 25 to 90 minutes. After 5-50minutes 100-fold excess (1-2 nmol) unlabeled lys-tRNA is added to thetranslation mixture. This serves to dilute out the labelleddansyl-lys-tRNA, allowing no further fluorescent reporter to beproduced. The GPI anchor, in addition to the reporter is observed byexciting the Trp residue in (i) with light of wavelength 280 nm andfollowing acceptor/donor (A/D) emission at appropriate wavelengths (˜340nm and ˜520 nm). Initially, FRET will be high, giving a high A/D output.As the processing of the reporter progresses the multimers are forcedapart by the addition of the GPI moiety, FRET is lost and the A/D ratiodecreases.

EXAMPLE 7

Protection of the Reporter From Promiscuous Modification

In order to configure the assays of Examples 1 to 5 not only to monitorchemical modification in defined systems but also to screen foractivities in complex environments (e.g. whole cell lysates) it may benecessary to protect the reporter molecule from proteolysis at sitesother than those engineered into the structure. The vast majority ofnaturally occurring enzymes will not recognise as substrates peptidescomprising D amino acids. The specificity of the reporter group will beincreased by limiting the use of L amino acids (recognised by enzymes)to residues involved in recognition by the protease. This will greatlyreduce the probability of unplanned proteolysis at additional sites byother proteases in, for example, a cell extract.

Proteins or peptides composed of D amino acids have a structure which isthe exact mirror image of that formed with L amino acids (Identificationof D-peptide ligands through mirror image phage display, Schumacher, T.N. M., Mayr, L. M., Minor, D. L. Jnr., Milhollen, M. A., Burgess, M. W.,Kim, P. S., Science (1996) 271 1854-1857). We therefore conclude that areporter designed in this way will have a structure of the form shown inthe diagram in FIG. 7.

1. A method of detecting or monitoring the activity of a proteaseenzyme, comprising the steps of: a) providing a first polypeptidebinding domain, and a second polypeptide binding domain, wherein i) atleast one of the binding domains comprises a site for protease digestionand wherein said site for protease digestion is located within saidfirst or second polypeptide binding domain; and ii) the first and secondbinding domains bind to each other such that a detectable signal isgenerated, and digestion of the binding domains at the site for proteasedigestion by the protease enzyme results in modulation of the binding ofthe polypeptides to each other and therefore of the detectable signal;b) allowing the binding domains to bind to each other and induce adetectable signal; c) contacting the binding domains with a proteaseenzyme; and d) detecting modulation of the detectable signal as a resultof the modulation of the binding of the binding domains.
 2. A method ofdetecting or monitoring the activity of a modulator of a proteaseenzyme, comprising the steps of: a) providing a first polypeptidebinding domain, and a second polypeptide binding domain, wherein i) atleast one of the binding domains comprises a site for protease digestionand wherein said site for protease digestion is located within saidfirst or second polypeptide binding domain; and ii) the first and secondbinding domains bind to each other such that a detectable signal isgenerated, and digestion of the polypeptides at the site for proteasedigestion by the protease enzyme results in modulation of the binding ofthe binding domains to each other and therefore of the detectablesignal; b) allowing the binding domains to bind to each other and inducea detectable signal; c) contacting the binding domains with a proteaseenzyme; d) detecting modulation of the detectable signal as a result ofthe modulation of the binding of the binding domains to determine areference signal modulation; e) contacting the binding domains with aprotease enzyme and a candidate modulator of the protease enzyme; and f)detecting modulation of the detectable signal as a result of themodulation of the binding of the binding domains to each other, andcomparing the modulation detected with the reference signal modulation.3. The method of claim 1 or claim 2, wherein at least one of the firstbinding domain or the second binding domain is labeled.
 4. The method ofclaim 3, wherein the label is a fluorescent label.
 5. The method ofclaim 3, wherein the first and second binding domains are associatedwith a first and second label, respectively.
 6. The method of claim 5,wherein the first label on the first binding domain is different fromthe label on the second binding domain.
 7. The method of claim 6,wherein said detectable signal is generated by an interaction betweenthe said first and second labels.
 8. The method of claim 6, wherein saidinteraction comprises energy transfer.
 9. The method of claim 1 or claim2, wherein said detectable signal is generated by a label associatedwith at least one of said first or second binding domains to diffuse insolution.
 10. The method of claim 1 or 2 wherein said digestioncomprises cleavage at a protease cleavable site.
 11. The method of claim1 or 2, wherein the binding domains are comprised in separatepolypeptides.
 12. The method of claim 1 or 2, wherein said first andsecond binding domains bind to each other to form a multimer; andwherein proteolysis is required for multimer association.